Accepted Manuscript Development of a heat mediated protein blotting method Jack O’Sullivan, Hilary E.M. McMahon PII:
S0003-2697(16)00051-8
DOI:
10.1016/j.ab.2016.01.026
Reference:
YABIO 12305
To appear in:
Analytical Biochemistry
Received Date: 18 January 2016 Revised Date:
29 January 2016
Accepted Date: 30 January 2016
Please cite this article as: J. O’Sullivan, H.E.M. McMahon, Development of a heat mediated protein blotting method, Analytical Biochemistry (2016), doi: 10.1016/j.ab.2016.01.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title: Development of a heat mediated protein blotting method
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Authors names: Jack O’Sullivan and Hilary E. M. McMahon*
Address of Authors:
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UCD School of Biomolecular and Biomedical Science, Conway Institute for Biomolecular and
Corresponding author:
Phone: +353 1 716 2196 Fax: +353 1 716 6456
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*
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Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.
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e-mail: [email protected]
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Abstract Western blotting is a significant tool employed for the detection of cell proteins. Large molecular weight proteins have proven a challenge to detect by western blotting, but the nature of proteins
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even of 100KDa can still present difficulties to detection. This work reports the development of a heat transfer method that is suitable for both low and high molecular weight proteins. The procedure involves the use of constant temperature at 78C in a dedicated heat transfer module. Through the
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use of this protocol the neuronal adaptor protein X11α (120KDa), which prior to this methodology was undetectable endogenously in the neuroblastoma cell line (N2a), was successfully detected in
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the N2a cell line. The procedure provides a reproducible protocol that can be adapted for other high molecular weight proteins and it provides the advantage that low molecular weight proteins are not
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Keywords
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sacrificed by the methodology.
Western blotting, heat transfer, high molecular weight.
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1. Introduction Electroblotting from a gel matrix after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to an immobilizing membrane such as nitrocellulose, nylon or Polyvinylidene
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fluoride (PVDF) is a significant tool for protein analysis [1-3]. The process relies on sufficient elution of the target protein from the gel matrix and adequate immobilisation on a detection membrane [4]. Success of transfer, however, can be hindered by the protein and its size, in general
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it becomes more difficult as the protein size increases [5, 6]. Proteins such as Titin, with a maximum subunit size over 4000kDa [7] are known to be difficult to electroblot, but smaller
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proteins over 100KDa can still pose problems. Standard electro transfer techniques are not always suitable for all proteins and this has led to the development of modified western blotting techniques [8, 9] or to proteolytically cleaving the protein of interest out of gels [10] and even physically
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disrupting the gel matrix to release the protein [11] to enhance the process of immunoblotting.
One of the factors limiting the elution of larger proteins from acrylamide gels is the pore size of the gel, low percentage acrylamide gels (3-6%) can aid the elution process, but such gels can be
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impractical to use and can have low resolution [9]. Irrespective the success of transfer is also influenced by the individual proteins composition and affinity for the receptor membrane [12].
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Approaches such as increased current and transfer times, have been used to overcome the difficulties seen with large proteins, but modifying conditions to maximize transfer efficiency of large proteins can lead to the loss of smaller proteins that may pass through the receiving membrane, decreasing or eliminating their detection. A more recent adaptation is the application of heat to make the gel more permeable to enable enhanced elution of high molecular weight proteins [5, 9]. By the application heat to western transfer Kurien and Scolfield [9] observed efficient
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transfer of both high and low molecular weight proteins
within 15mins, their methodology
provided for an efficient and rapid protein transfer method.
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During the course of this work with a neuronal adaptor protein, X11α, a 120KDa protein, we observed difficulties with its detection by standard western blotting techniques. Added to the difficulties with its detection is that the protein has low expression levels in the cell line employed,
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the neuroblastoma cell line (N2a). Past studies on this protein within the N2a cell line have generally overexpressed the protein by transfection to enable detection by standard Western blotting
protocols were
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procedures [13]. Our work required its detection without transfection, as a consequence various employed to detect X11α by western blot, including the short heat transfer
technique of Kurien and Scolfield [9], but these failed to enable reproducible detection of the protein. Nonetheless, adaptation of the protocol of Kurien and Scolfield [9] and alteration of the effective and reproducible detection of the low levels of
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western blotting apparatus facilitated
endogenous X11α in N2a cells. In this work we describe a technique based on heat transfer that can
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be adapted to improve detection of other large molecular weight proteins
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2. Materials and Methods 2.1 Reagents and Antibodies.
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Protran 0.45µm Nitrocellulose membrane was from Fisher Scientific. Primary antibody used for X11α detection was a rabbit polyclonal IgG (H-256) obtained from Santa Cruz Biotechnology Inc. Secondary HRP-conjugated Goat Anti-Rabbit IgG antibody was from Millipore. Rat cerebrum
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lysate was from BD Biosciences. Broad range pre-stained molecular weight markers were from
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New England Biolabs. All other reagents were supplied by Sigma-Aldrich.
2.2 Cell culture.
N2a cells were grown in Dulbecco’s Modified Essential Medium (DMEM) supplemented with 10% (v/v) fetal calf serum and 10mM penicillin-streptomycin. The neuroblastoma cell line overexpressing the normal prion protein (PrPC) N2a22L20Cr, which was reported previously [14]
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was grown in normal N2a media containing 300µg/ml geneticin. Cells were lysed in cold RIPASDS lysis buffer (LB) (0.5% (w/v) Sodium Deoxycholate, 150mM NaCl, 1%(v/v) NP40, 0.1% (w/v) SDS and 50mM Tris-HCl pH 6.8, 1µg/ml pepstatin, 1µg/ml leupeptin, 2mM EDTA). The
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total protein concentration was measured using the bicinchoninic acid protein assay kit (BCA assay)
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(Sigma). All samples were made up to 20µl in loading dye (0.06M Tris-HCl pH 6.8, 2% (w/v) SDS, 5% (w/v), 100mM DTT, 10% (v/v) glycerol, 0.12mg/ml bromophenol blue), heated to 100°C for 5 min and analysed by 8% SDS-PAGE. Following electrophoresis western blotting was carried out as indicated in the text.
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2.3 Standard western blotting methods and conditions Standard western blotting was carried out using the buffers indicated. Western transfers were carried out at room temperature (RT), with ice blocks for cooling as per manufacturer instruction, at
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400Ma for 1h onto nitrocellulose membrane as indicated. Protein was detected by incubating immunoblots with the antibodies indicated followed by a horseradish peroxidase (HRP) secondary
2.4 Long Heat Transfer Western blotting method
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antibody and developed by enhanced chemiluminescence (ECL).
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The method of Kurien and Schofield [9], had advised the use of a western module from Hoeffer Scientific Instruments which could circulate heated water around the bottom of the tank, our adapted technique identified that a stronger control of temperature was needed. For this we adapted a standard western blotting apparatus (Figure 1). An electric mixer was attached to the base of the
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transfer apparatus lid to allow for adequate heat transfer in the transfer buffer. A port was generated for a thermometer to monitor the temperature inside the tank during the transfer process. The tank was placed inside a temperature controlled water bath set to 78°C for the duration of the experiment
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and water within the water bath was constantly circulated. Heat transfer was run using Heat
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Transfer buffer (HTB) (25mM Tris, 192mM glycine).
The following outlines the conditions required for the long heat transfer westernblot. In preparation and before carrying out gel electrophoresis a water bath was set to 78°C, the Heat Transfer (HT) Buffer was then brought to 78°C in the same water bath. The western sponges and filter paper were soaked in 1L of Heat Transfer Buffer but at room temperature (RT). The nitrocellulose membrane was activated by immersion in cold HT Buffer containing 20% methanol for 1h. After SDS-PAGE
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electrophoresis, the gel (0.75mm width) was removed carefully and immersed in HT Buffer at RT for 10minutes. This reduces swelling during transfer and distortion of the gel. For thicker or higher percentage acrylamide gels extend equilibration time. The transfer cassettes with the sponges, filter
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papers, membrane and gel were assembled together at room temperature. The HT Buffer pre-heated to 78°C was poured into the transfer tank and the cassette was immerse gently. The transfer was run at constant 400Ma for 1.5h with constant stirring, this stirring was achieved by the mechanical
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mixer (figure 1). During transfer the temperature of the water bath was periodically checked, as was the temperature maintained inside the tank. The temperature was maintained at 78°C without
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stopping transfer. Using a current over 400Ma will cause the temperature inside the tank to rise steadily as excess heat is generated, using a constant current of 400Ma should allow the temperature to remain constant throughout the run. At the end of the transfer protein was detected as per the
3 Results
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standard western blotting procedure.
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3.1 Detection of X11α by standard western blotting procedure During our work on X11α it was noted that standard western blotting techniques did not allow for
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the detection of endogenous X11α in the N2a cell line irrespective of varying buffer conditions (figure 2 A, B and C). For this work we employed two cell lines the N2a cell line and a transfected cell line N2a22L20Cr. These cell lines were employed in our a recent study [14] to examine the effect of the normal prion protein (PrPC) on the expression of X11α. The N2a22L20Cr cell line stably overexpresses PrPC, this overexpression resulted in increased expression of X11α in the N2a cell line which we could detect by standard western blotting, but full length X11α could not be
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detected easily in N2a cells. As a result we hypothesised that if a sensitive western blotting protocol
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was available then endogenous X11α in the N2a cell line would be possible.
X11α is a 120KDa protein which is detectable in rat lysate by standard western blotting (figure 2 lane 1). In the N2a cell line X11α can be detected as two bands [14], and its full length size is 120KDa. The different banding seen with X11α has been proposed to be due differences in
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phosphorylation[15] or to do with protein fragmentation [16]. To establish conditions for transfer a
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number of buffers were tested for their ability to transfer the protein out of the gel. The most commonly used buffer used in western blotting is the Tris-Glycine buffer (25mM Tris, 192mM Glycine, 20% methanol) first used by Towbin et al., [17]. Using this original buffer only slight levels of full length X11α were observed in the N2a22L20Cr cell line (Figure 2 lane 2), whilst in N2a cells only faint levels of the lower band were detected (figure 2 lane 3). Through SiRNA
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knockdown of X11α, both bands as observed in lane 2 were identified as X11α (data not shown). Initial attempts to increase detection looked at modification of the standard buffer. The methanol in the buffer causes stripping of complex SDS from the protein samples [18] and is used to facilitate
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protein transfer. However it can partially fix proteins into gels reducing efficiency of transfer of
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large proteins [12, 19], as a consequence we reduced the level of methanol to 10% (figure 2B). Reducing the methanol increased detection of the X11α upper band in the N2a22L20Cr cell line, but failed to increase detection in N2a cells (figure 2B, lane 4 and 5).
The final parameter adjusted alongside methanol was SDS content, which is known to affect release of protein from acrylamide gels [20]. Bolt and Mahoney observed SDS to significantly reduce the transfer of high molecular weight proteins [12] especially as levels increased to 0.1%. Both
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methanol and SDS were removed from the transfer buffer, however this did not significantly improve detection (figure 2C, lanes 6 and 7), although higher detection of the upper band was seen
3.2 Development of a heat transfer unit for western blotting.
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in N2a22L20Cr cells (figure 2C lane 6).
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As the above conditions failed to improve a reproducible detection of X11α we employed the procedure of Kurien and Schofield’s [9], but it was still not possible to detect endogenous X11α
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levels (data not shown). When following Kurien and Schofield’s methods several observations were made. Using 40V for transfer resulted in the applied current approaching the limit of what most standard power supplies can produce (500Ma). This high current caused considerable heat to be generated, causing the temperature in the transfer tank to rise steadily during the run to above 70°C. In this work, it was noticed that if the temperature rises above 80°C, the gel can start to become
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damaged from excess heat (data not shown). It was also observed that using only 15min transfer time, as outlined in the method of Kurien and Schofield [9] in combination with protein loads >30µg left considerable detectable levels of protein in the gel after staining with Coomassie Blue
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solution (data not shown).
The method of Kurien and Schofield [9], had advised the use of western module from Hoeffer Scientific Instruments which could circulate water around the bottom of the tank. For our work the Mini Trans-Blot® (Biorad) was used, and a number of adaptations were made to the unit to allow for continuous heat control during the protein transfer (figure 1). Utilising a set current of 400Ma and a water bath to submerse the tank allowed for a greater control of temperature, allowing a set temperature of 78°C to be maintained throughout the transfer. The heat of transfer was raised to
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78°C rather than that of 70°C as published by Kurien and Schofield [9]. The higher heat gave more successful transfer than blots run at 70°C under the same conditions (data not shown). A mechanical mixer/electric stirrer affixed to the lid of the tank was used instead of the more
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traditional magnetic stirring. This was required as the western unit was immersed in a heated water bath. Through optimisation we identified that the Heat Transfer buffer 25mM Tris, 192mM glycine, without MeOH or SDS, allowed for suitable transfer of X11α under heat conditions (figure 2D
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lanes 8 and 9). Under these conditions transfer of the upper band was substantially increased, and
extraction of X11α from the gel matrix.
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endogenous X11α was now detectable in the N2a cell line. These conditions enabled reproducible
3.3 Effect of the modified heat transfer technique on the transfer of other proteins. To demonstrate the applicability of this heat transfer protocol to other proteins, we analyzed the
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transfer of pre-strained molecular weight markers, by standard and by our heat transfer protocol (figure 3). These markers contain a number of different proteins and all were transferred by the heat transfer (HT) protocol to the nitrocellulose membrane across better than the standard transfer (ST)
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(Figure 3, compare lanes 1 and 2). The molecular weight markers could be detected as low as
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17kDa up to 175kDa indicating this method could allow for the transfer of both high and low molecular weight proteins. The heat transfer was notably more successful at transfer of the protein markers than standard transfer.
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4. Discussion Western blotting of proteins has evolved significantly since its origins when used by Towbin et al.,
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[17]. The process has been complicated by the nature of proteins influencing the success of transfer [12]. In particular high molecular weight proteins have received much interest, as they have led to the greatest challenge when western blotting [5, 9, 12], leading to extended western blotting times
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up to 21 hours [21]. In this work we have adapted a process of heat to the western blotting of X11α, which was difficult to elute by standard western blotting procedures. Kurien and Scolfield [5],
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originally developed a 15min heat transfer for western blotting. This work describes the evolution of a long heat transfer process of 1.5h that can be adapted to both small and high molecular weight proteins.
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Until recently X11α had not been detected endogenously in N2a cells, and its detection was facilitated by the development of the long heat transfer protocol [14] reported here. In the process of improving detection of X11α by the standard western transfer process, we altered methanol and
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SDS levels, but neither led to significant improvement in the proteins detection. In standard western blotting methanol aids in the removal of complex SDS from the protein samples [18], prevents gel
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swelling and distortion during transfer and can aid binding to nitrocellulose membranes[9]. However, methanol can fix larger proteins in the gel resulting in a decreased efficiency of their elution [12, 19], removing methanol had no impact on our detection of X11α by standard transfer. A common modification of the Tris Glycine buffer used by Towbin et al., [17] involves the addition of 0.1% SDS. The SDS can increase the solubility of proteins [20] and permit elution from the gel. However, in contradiction, a drawback is that 0.1% SDS has been shown to hinder the transfer of
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larger proteins [12]. For X11α eliminating SDS from standard western blotting had negligible effect
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on increasing its efficiency of transfer.
Kurien and Scofield introduced a rapid western transfer based on the use of heat to remove proteins from the gel matrix [5]. While the method was demonstrated to be effective at rapidly transferring proteins from different thickness and % acrylamide gels within 15min, the protocol could not be
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used for X11α. In this work it was identified that extended transfer time was required up to 1.5h to
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visualise X11α in N2a cells, the protocol allowed for transfer of proteins out of the gel even when large protein loads were used. Although our protocol takes away the advantage of the rapid element of the Kurien and Scofield heat method (2002), it facilitated in the detection of X11α within N2a cells which would otherwise have remained undetected. In the past procedures directed towards high molecular weight proteins have been found to have limitations in that they are detected at the
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expense of smaller proteins. Wang et al., (1989) observed that conditions optimised for proteins >200kDa resulted in the loss of detection of proteins smaller than 40kDa. In this work we observed both large and small proteins to be efficiently transferred and retained on the receiving membrane
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when our heat transfer protocol is used.
5. Conclusion
The method presented here advances on the original heat transfer procedure of Kurien and Scofield [5]. Whilst our protocol sacrifices the rapid element of the Kurien and Scofield’s procedure [5], their 15min transfer, this was necessary to detect endogenous levels of X11α in N2a cells. Our protocol was successful in the transfer of both high and low molecular weight proteins, and it facilitated the detection of a protein which would otherwise not have been appropriately detected.
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The procedure outlined complements that of Kurien and Scofield’s [5], where their short procedure may not work for a given protein this extended procedure could be adopted. This method could be used to detect low levels of other endogenous protein in samples that have previously been found
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difficult to western blot.
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6. Acknowledgements
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J.O’S was supported by Science Foundation Ireland (08/RFP/BIC1151),
7. References
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[2] J.L. Luque-Garcia, G. Zhou, D.S. Spellman, T.T. Sun, T.A. Neubert, Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting, Molecular & cellular proteomics : MCP, 7 (2008) 308-314.
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[3] A. Shevchenko, M. Wilm, O. Vorm, M. Mann, Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels, Analytical chemistry, 68 (1996) 850-858.
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[4] Z. Jaunmuktane, S. Mead, M. Ellis, J.D. Wadsworth, A.J. Nicoll, J. Kenny, F. Launchbury, J. Linehan, A. Richard-Loendt, A.S. Walker, P. Rudge, J. Collinge, S. Brandner, Evidence for human transmission of amyloid-beta pathology and cerebral amyloid angiopathy, Nature, 525 (2015) 247250. [5] B.T. Kurien, R.H. Scofield, Heat-mediated, ultra-rapid electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes, J Immunol Methods, 266 (2002) 127-133.
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[6] T. Otter, S.M. King, G.B. Witman, A two-step procedure for efficient electrotransfer of both high-molecular-weight (greater than 400,000) and low-molecular-weight (less than 20,000) proteins, Analytical biochemistry, 162 (1987) 370-377.
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[7] M.L. Bang, T. Centner, F. Fornoff, A.J. Geach, M. Gotthardt, M. McNabb, C.C. Witt, D. Labeit, C.C. Gregorio, H. Granzier, S. Labeit, The complete gene sequence of titin, expression of an unusual approximately 700-kDa titin isoform, and its interaction with obscurin identify a novel Z-
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[8] C.M. Warren, P.R. Krzesinski, M.L. Greaser, Vertical agarose gel electrophoresis and
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electroblotting of high-molecular-weight proteins, Electrophoresis, 24 (2003) 1695-1702. [9] B.T. Kurien, R.H. Scofield, Ultrarapid electrophoretic transfer of high and low molecular weight proteins using heat, Methods Mol Biol, 536 (2009) 181-190.
[10] W. Gibson, Protease-facilitated transfer of high-molecular-weight proteins during
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electrotransfer to nitrocellulose, Analytical biochemistry, 118 (1981) 1-3. [11] J. Renart, J. Reiser, G.R. Stark, Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure, Proc
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[12] M.W. Bolt, P.A. Mahoney, High-efficiency blotting of proteins of diverse sizes following
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sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Analytical biochemistry, 247 (1997) 185-192.
[13] A. Sumioka, Y. Saito, M. Sakuma, Y. Araki, T. Yamamoto, T. Suzuki, The X11L/X11beta/MINT2 and X11L2/X11gamma/MINT3 scaffold proteins shuttle between the nucleus and cytoplasm, Experimental cell research, 314 (2008) 1155-1162.
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[14] J. O'Sullivan, E. Comerford, W. Rachidi, M. Scott, N.M. Hooper, H.E. McMahon, The effects of the cellular and infectious prion protein on the neuronal adaptor protein X11alpha, Biochim Biophys Acta., 1850 (2015) 2213-2221. doi: 2210.1016/j.bbagen.2015.2208.2010.
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[15] J. Chaufty, S.E. Sullivan, A. Ho, Intracellular amyloid precursor protein sorting and amyloidbeta secretion are regulated by Src-mediated phosphorylation of Mint2, J Neurosci, 32 (2012) 96139625.
mouse hippocampus, Eur J Neurosci, 12 (2000) 3067-3072.
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[16] M. Okamoto, T. Matsuyama, M. Sugita, Ultrastructural localization of mint1 at synapses in
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[17] H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc Natl Acad Sci U S A, 76 (1979) 4350-4354.
[18] J. Mozdzanowski, D.W. Speicher, Microsequence analysis of electroblotted proteins. I.
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Comparison of electroblotting recoveries using different types of PVDF membranes, Analytical biochemistry, 207 (1992) 11-18.
[19] U. Beisiegel, Protein blotting, Electrophoresis, 7 (1986) 1-18.
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[20] H. Towbin, J. Gordon, Immunoblotting and dot immunobinding--current status and outlook, J Immunol Methods, 72 (1984) 313-340.
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[21] P.F. Erickson, L.N. Minier, R.S. Lasher, Quantitative electrophoretic transfer of polypeptides from SDS polyacrylamide gels to nitrocellulose sheets: a method for their re-use in immunoautoradiographic detection of antigens, J Immunol Methods, 51 (1982) 241-249.
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Figure legends Figure 1. Diagram of apparatus used for heat transfer method Diagram showing set up of heat
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method including waterbath, mechanical mixing and circulation of water in waterbath via pump.
Figure 2. Comparison of heat western transfer to standard western transfer with modified buffers 20µg of cell line lysates of N2a and N2a22L20Cr cells were analysed by 8% SDS-PAGE
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followed by immunoblotting with X11α antibody. Standard western conditions (1h at 400mA, Room temperature) were used for A-C with a Tris-Glycine buffer (25mM Tris, 192mM Glycine)
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containing the level of methanol (MeOH) and SDS indicated. (D) Western was performed in a TrisGlycine buffer (25mM Tris, 192mM Glycine) without MeOH or SDS. Transfer was carried out at 78C for 1.5h at 400mA. Lanes 1 of A contains 3µg Rat Cerebrum Lysate (BD Biosciences). Protein size is indicated on the left of panels in kDa. Results are representative of three individual
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experiments. Arrow indicates location of full length X11α.
Figure 3. Immobilisation of molecular weight markers using standard and heat western transfer 6µl of commercial pre-strained molecular weight markers were analysed by 8% SDS-
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PAGE (Lanes 1 and 2). Lane 1 sample was transferred onto nitrocellulose by Heat transfer (HT) in
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25mM Tris, 192mM Glycine for 1.5h at 400mA. Lane 2 sample was transferred by standard transfer (1h at 400Ma at RT) with a standard Tris-Glycine buffer (25mM Tris, 192mM Glycine, 20% MeOH, 0.1% SDS). Membranes were imaged simultaneously using white light trans-luminescence. Protein size is indicated on the left of panels in kDa, what these proteins are is indicated on the right. Where more than one entity facilitates the protein size this is a fusion of proteins. Results are representative of three individual experiments.
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Figure 1
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Lane 175.0 -
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Figure 2
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_____________________________________
Transfer conditions
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N2a 22L20Cr
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Figure 3
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Lysozyme HT
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S0003-2697(16)00051-8
DOI:
10.1016/j.ab.2016.01.026
Reference:
YABIO 12305
To appear in:
Analytical Biochemistry
Received Date: 18 January 2016 Revised Date:
29 January 2016
Accepted Date: 30 January 2016
Please cite this article as: J. O’Sullivan, H.E.M. McMahon, Development of a heat mediated protein blotting method, Analytical Biochemistry (2016), doi: 10.1016/j.ab.2016.01.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title: Development of a heat mediated protein blotting method
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Authors names: Jack O’Sullivan and Hilary E. M. McMahon*
Address of Authors:
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UCD School of Biomolecular and Biomedical Science, Conway Institute for Biomolecular and
Corresponding author:
Phone: +353 1 716 2196 Fax: +353 1 716 6456
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*
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Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.
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e-mail: [email protected]
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Abstract Western blotting is a significant tool employed for the detection of cell proteins. Large molecular weight proteins have proven a challenge to detect by western blotting, but the nature of proteins
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even of 100KDa can still present difficulties to detection. This work reports the development of a heat transfer method that is suitable for both low and high molecular weight proteins. The procedure involves the use of constant temperature at 78C in a dedicated heat transfer module. Through the
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use of this protocol the neuronal adaptor protein X11α (120KDa), which prior to this methodology was undetectable endogenously in the neuroblastoma cell line (N2a), was successfully detected in
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the N2a cell line. The procedure provides a reproducible protocol that can be adapted for other high molecular weight proteins and it provides the advantage that low molecular weight proteins are not
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Keywords
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sacrificed by the methodology.
Western blotting, heat transfer, high molecular weight.
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1. Introduction Electroblotting from a gel matrix after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to an immobilizing membrane such as nitrocellulose, nylon or Polyvinylidene
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fluoride (PVDF) is a significant tool for protein analysis [1-3]. The process relies on sufficient elution of the target protein from the gel matrix and adequate immobilisation on a detection membrane [4]. Success of transfer, however, can be hindered by the protein and its size, in general
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it becomes more difficult as the protein size increases [5, 6]. Proteins such as Titin, with a maximum subunit size over 4000kDa [7] are known to be difficult to electroblot, but smaller
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proteins over 100KDa can still pose problems. Standard electro transfer techniques are not always suitable for all proteins and this has led to the development of modified western blotting techniques [8, 9] or to proteolytically cleaving the protein of interest out of gels [10] and even physically
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disrupting the gel matrix to release the protein [11] to enhance the process of immunoblotting.
One of the factors limiting the elution of larger proteins from acrylamide gels is the pore size of the gel, low percentage acrylamide gels (3-6%) can aid the elution process, but such gels can be
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impractical to use and can have low resolution [9]. Irrespective the success of transfer is also influenced by the individual proteins composition and affinity for the receptor membrane [12].
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Approaches such as increased current and transfer times, have been used to overcome the difficulties seen with large proteins, but modifying conditions to maximize transfer efficiency of large proteins can lead to the loss of smaller proteins that may pass through the receiving membrane, decreasing or eliminating their detection. A more recent adaptation is the application of heat to make the gel more permeable to enable enhanced elution of high molecular weight proteins [5, 9]. By the application heat to western transfer Kurien and Scolfield [9] observed efficient
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transfer of both high and low molecular weight proteins
within 15mins, their methodology
provided for an efficient and rapid protein transfer method.
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During the course of this work with a neuronal adaptor protein, X11α, a 120KDa protein, we observed difficulties with its detection by standard western blotting techniques. Added to the difficulties with its detection is that the protein has low expression levels in the cell line employed,
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the neuroblastoma cell line (N2a). Past studies on this protein within the N2a cell line have generally overexpressed the protein by transfection to enable detection by standard Western blotting
protocols were
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procedures [13]. Our work required its detection without transfection, as a consequence various employed to detect X11α by western blot, including the short heat transfer
technique of Kurien and Scolfield [9], but these failed to enable reproducible detection of the protein. Nonetheless, adaptation of the protocol of Kurien and Scolfield [9] and alteration of the effective and reproducible detection of the low levels of
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western blotting apparatus facilitated
endogenous X11α in N2a cells. In this work we describe a technique based on heat transfer that can
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be adapted to improve detection of other large molecular weight proteins
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2. Materials and Methods 2.1 Reagents and Antibodies.
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Protran 0.45µm Nitrocellulose membrane was from Fisher Scientific. Primary antibody used for X11α detection was a rabbit polyclonal IgG (H-256) obtained from Santa Cruz Biotechnology Inc. Secondary HRP-conjugated Goat Anti-Rabbit IgG antibody was from Millipore. Rat cerebrum
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lysate was from BD Biosciences. Broad range pre-stained molecular weight markers were from
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New England Biolabs. All other reagents were supplied by Sigma-Aldrich.
2.2 Cell culture.
N2a cells were grown in Dulbecco’s Modified Essential Medium (DMEM) supplemented with 10% (v/v) fetal calf serum and 10mM penicillin-streptomycin. The neuroblastoma cell line overexpressing the normal prion protein (PrPC) N2a22L20Cr, which was reported previously [14]
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was grown in normal N2a media containing 300µg/ml geneticin. Cells were lysed in cold RIPASDS lysis buffer (LB) (0.5% (w/v) Sodium Deoxycholate, 150mM NaCl, 1%(v/v) NP40, 0.1% (w/v) SDS and 50mM Tris-HCl pH 6.8, 1µg/ml pepstatin, 1µg/ml leupeptin, 2mM EDTA). The
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total protein concentration was measured using the bicinchoninic acid protein assay kit (BCA assay)
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(Sigma). All samples were made up to 20µl in loading dye (0.06M Tris-HCl pH 6.8, 2% (w/v) SDS, 5% (w/v), 100mM DTT, 10% (v/v) glycerol, 0.12mg/ml bromophenol blue), heated to 100°C for 5 min and analysed by 8% SDS-PAGE. Following electrophoresis western blotting was carried out as indicated in the text.
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2.3 Standard western blotting methods and conditions Standard western blotting was carried out using the buffers indicated. Western transfers were carried out at room temperature (RT), with ice blocks for cooling as per manufacturer instruction, at
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400Ma for 1h onto nitrocellulose membrane as indicated. Protein was detected by incubating immunoblots with the antibodies indicated followed by a horseradish peroxidase (HRP) secondary
2.4 Long Heat Transfer Western blotting method
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antibody and developed by enhanced chemiluminescence (ECL).
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The method of Kurien and Schofield [9], had advised the use of a western module from Hoeffer Scientific Instruments which could circulate heated water around the bottom of the tank, our adapted technique identified that a stronger control of temperature was needed. For this we adapted a standard western blotting apparatus (Figure 1). An electric mixer was attached to the base of the
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transfer apparatus lid to allow for adequate heat transfer in the transfer buffer. A port was generated for a thermometer to monitor the temperature inside the tank during the transfer process. The tank was placed inside a temperature controlled water bath set to 78°C for the duration of the experiment
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and water within the water bath was constantly circulated. Heat transfer was run using Heat
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Transfer buffer (HTB) (25mM Tris, 192mM glycine).
The following outlines the conditions required for the long heat transfer westernblot. In preparation and before carrying out gel electrophoresis a water bath was set to 78°C, the Heat Transfer (HT) Buffer was then brought to 78°C in the same water bath. The western sponges and filter paper were soaked in 1L of Heat Transfer Buffer but at room temperature (RT). The nitrocellulose membrane was activated by immersion in cold HT Buffer containing 20% methanol for 1h. After SDS-PAGE
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electrophoresis, the gel (0.75mm width) was removed carefully and immersed in HT Buffer at RT for 10minutes. This reduces swelling during transfer and distortion of the gel. For thicker or higher percentage acrylamide gels extend equilibration time. The transfer cassettes with the sponges, filter
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papers, membrane and gel were assembled together at room temperature. The HT Buffer pre-heated to 78°C was poured into the transfer tank and the cassette was immerse gently. The transfer was run at constant 400Ma for 1.5h with constant stirring, this stirring was achieved by the mechanical
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mixer (figure 1). During transfer the temperature of the water bath was periodically checked, as was the temperature maintained inside the tank. The temperature was maintained at 78°C without
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stopping transfer. Using a current over 400Ma will cause the temperature inside the tank to rise steadily as excess heat is generated, using a constant current of 400Ma should allow the temperature to remain constant throughout the run. At the end of the transfer protein was detected as per the
3 Results
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standard western blotting procedure.
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3.1 Detection of X11α by standard western blotting procedure During our work on X11α it was noted that standard western blotting techniques did not allow for
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the detection of endogenous X11α in the N2a cell line irrespective of varying buffer conditions (figure 2 A, B and C). For this work we employed two cell lines the N2a cell line and a transfected cell line N2a22L20Cr. These cell lines were employed in our a recent study [14] to examine the effect of the normal prion protein (PrPC) on the expression of X11α. The N2a22L20Cr cell line stably overexpresses PrPC, this overexpression resulted in increased expression of X11α in the N2a cell line which we could detect by standard western blotting, but full length X11α could not be
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detected easily in N2a cells. As a result we hypothesised that if a sensitive western blotting protocol
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was available then endogenous X11α in the N2a cell line would be possible.
X11α is a 120KDa protein which is detectable in rat lysate by standard western blotting (figure 2 lane 1). In the N2a cell line X11α can be detected as two bands [14], and its full length size is 120KDa. The different banding seen with X11α has been proposed to be due differences in
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phosphorylation[15] or to do with protein fragmentation [16]. To establish conditions for transfer a
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number of buffers were tested for their ability to transfer the protein out of the gel. The most commonly used buffer used in western blotting is the Tris-Glycine buffer (25mM Tris, 192mM Glycine, 20% methanol) first used by Towbin et al., [17]. Using this original buffer only slight levels of full length X11α were observed in the N2a22L20Cr cell line (Figure 2 lane 2), whilst in N2a cells only faint levels of the lower band were detected (figure 2 lane 3). Through SiRNA
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knockdown of X11α, both bands as observed in lane 2 were identified as X11α (data not shown). Initial attempts to increase detection looked at modification of the standard buffer. The methanol in the buffer causes stripping of complex SDS from the protein samples [18] and is used to facilitate
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protein transfer. However it can partially fix proteins into gels reducing efficiency of transfer of
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large proteins [12, 19], as a consequence we reduced the level of methanol to 10% (figure 2B). Reducing the methanol increased detection of the X11α upper band in the N2a22L20Cr cell line, but failed to increase detection in N2a cells (figure 2B, lane 4 and 5).
The final parameter adjusted alongside methanol was SDS content, which is known to affect release of protein from acrylamide gels [20]. Bolt and Mahoney observed SDS to significantly reduce the transfer of high molecular weight proteins [12] especially as levels increased to 0.1%. Both
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methanol and SDS were removed from the transfer buffer, however this did not significantly improve detection (figure 2C, lanes 6 and 7), although higher detection of the upper band was seen
3.2 Development of a heat transfer unit for western blotting.
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in N2a22L20Cr cells (figure 2C lane 6).
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As the above conditions failed to improve a reproducible detection of X11α we employed the procedure of Kurien and Schofield’s [9], but it was still not possible to detect endogenous X11α
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levels (data not shown). When following Kurien and Schofield’s methods several observations were made. Using 40V for transfer resulted in the applied current approaching the limit of what most standard power supplies can produce (500Ma). This high current caused considerable heat to be generated, causing the temperature in the transfer tank to rise steadily during the run to above 70°C. In this work, it was noticed that if the temperature rises above 80°C, the gel can start to become
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damaged from excess heat (data not shown). It was also observed that using only 15min transfer time, as outlined in the method of Kurien and Schofield [9] in combination with protein loads >30µg left considerable detectable levels of protein in the gel after staining with Coomassie Blue
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solution (data not shown).
The method of Kurien and Schofield [9], had advised the use of western module from Hoeffer Scientific Instruments which could circulate water around the bottom of the tank. For our work the Mini Trans-Blot® (Biorad) was used, and a number of adaptations were made to the unit to allow for continuous heat control during the protein transfer (figure 1). Utilising a set current of 400Ma and a water bath to submerse the tank allowed for a greater control of temperature, allowing a set temperature of 78°C to be maintained throughout the transfer. The heat of transfer was raised to
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78°C rather than that of 70°C as published by Kurien and Schofield [9]. The higher heat gave more successful transfer than blots run at 70°C under the same conditions (data not shown). A mechanical mixer/electric stirrer affixed to the lid of the tank was used instead of the more
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traditional magnetic stirring. This was required as the western unit was immersed in a heated water bath. Through optimisation we identified that the Heat Transfer buffer 25mM Tris, 192mM glycine, without MeOH or SDS, allowed for suitable transfer of X11α under heat conditions (figure 2D
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lanes 8 and 9). Under these conditions transfer of the upper band was substantially increased, and
extraction of X11α from the gel matrix.
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endogenous X11α was now detectable in the N2a cell line. These conditions enabled reproducible
3.3 Effect of the modified heat transfer technique on the transfer of other proteins. To demonstrate the applicability of this heat transfer protocol to other proteins, we analyzed the
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transfer of pre-strained molecular weight markers, by standard and by our heat transfer protocol (figure 3). These markers contain a number of different proteins and all were transferred by the heat transfer (HT) protocol to the nitrocellulose membrane across better than the standard transfer (ST)
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(Figure 3, compare lanes 1 and 2). The molecular weight markers could be detected as low as
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17kDa up to 175kDa indicating this method could allow for the transfer of both high and low molecular weight proteins. The heat transfer was notably more successful at transfer of the protein markers than standard transfer.
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4. Discussion Western blotting of proteins has evolved significantly since its origins when used by Towbin et al.,
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[17]. The process has been complicated by the nature of proteins influencing the success of transfer [12]. In particular high molecular weight proteins have received much interest, as they have led to the greatest challenge when western blotting [5, 9, 12], leading to extended western blotting times
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up to 21 hours [21]. In this work we have adapted a process of heat to the western blotting of X11α, which was difficult to elute by standard western blotting procedures. Kurien and Scolfield [5],
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originally developed a 15min heat transfer for western blotting. This work describes the evolution of a long heat transfer process of 1.5h that can be adapted to both small and high molecular weight proteins.
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Until recently X11α had not been detected endogenously in N2a cells, and its detection was facilitated by the development of the long heat transfer protocol [14] reported here. In the process of improving detection of X11α by the standard western transfer process, we altered methanol and
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SDS levels, but neither led to significant improvement in the proteins detection. In standard western blotting methanol aids in the removal of complex SDS from the protein samples [18], prevents gel
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swelling and distortion during transfer and can aid binding to nitrocellulose membranes[9]. However, methanol can fix larger proteins in the gel resulting in a decreased efficiency of their elution [12, 19], removing methanol had no impact on our detection of X11α by standard transfer. A common modification of the Tris Glycine buffer used by Towbin et al., [17] involves the addition of 0.1% SDS. The SDS can increase the solubility of proteins [20] and permit elution from the gel. However, in contradiction, a drawback is that 0.1% SDS has been shown to hinder the transfer of
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larger proteins [12]. For X11α eliminating SDS from standard western blotting had negligible effect
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on increasing its efficiency of transfer.
Kurien and Scofield introduced a rapid western transfer based on the use of heat to remove proteins from the gel matrix [5]. While the method was demonstrated to be effective at rapidly transferring proteins from different thickness and % acrylamide gels within 15min, the protocol could not be
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used for X11α. In this work it was identified that extended transfer time was required up to 1.5h to
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visualise X11α in N2a cells, the protocol allowed for transfer of proteins out of the gel even when large protein loads were used. Although our protocol takes away the advantage of the rapid element of the Kurien and Scofield heat method (2002), it facilitated in the detection of X11α within N2a cells which would otherwise have remained undetected. In the past procedures directed towards high molecular weight proteins have been found to have limitations in that they are detected at the
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expense of smaller proteins. Wang et al., (1989) observed that conditions optimised for proteins >200kDa resulted in the loss of detection of proteins smaller than 40kDa. In this work we observed both large and small proteins to be efficiently transferred and retained on the receiving membrane
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when our heat transfer protocol is used.
5. Conclusion
The method presented here advances on the original heat transfer procedure of Kurien and Scofield [5]. Whilst our protocol sacrifices the rapid element of the Kurien and Scofield’s procedure [5], their 15min transfer, this was necessary to detect endogenous levels of X11α in N2a cells. Our protocol was successful in the transfer of both high and low molecular weight proteins, and it facilitated the detection of a protein which would otherwise not have been appropriately detected.
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The procedure outlined complements that of Kurien and Scofield’s [5], where their short procedure may not work for a given protein this extended procedure could be adopted. This method could be used to detect low levels of other endogenous protein in samples that have previously been found
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difficult to western blot.
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6. Acknowledgements
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J.O’S was supported by Science Foundation Ireland (08/RFP/BIC1151),
7. References
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[2] J.L. Luque-Garcia, G. Zhou, D.S. Spellman, T.T. Sun, T.A. Neubert, Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting, Molecular & cellular proteomics : MCP, 7 (2008) 308-314.
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[3] A. Shevchenko, M. Wilm, O. Vorm, M. Mann, Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels, Analytical chemistry, 68 (1996) 850-858.
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[4] Z. Jaunmuktane, S. Mead, M. Ellis, J.D. Wadsworth, A.J. Nicoll, J. Kenny, F. Launchbury, J. Linehan, A. Richard-Loendt, A.S. Walker, P. Rudge, J. Collinge, S. Brandner, Evidence for human transmission of amyloid-beta pathology and cerebral amyloid angiopathy, Nature, 525 (2015) 247250. [5] B.T. Kurien, R.H. Scofield, Heat-mediated, ultra-rapid electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes, J Immunol Methods, 266 (2002) 127-133.
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[6] T. Otter, S.M. King, G.B. Witman, A two-step procedure for efficient electrotransfer of both high-molecular-weight (greater than 400,000) and low-molecular-weight (less than 20,000) proteins, Analytical biochemistry, 162 (1987) 370-377.
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[7] M.L. Bang, T. Centner, F. Fornoff, A.J. Geach, M. Gotthardt, M. McNabb, C.C. Witt, D. Labeit, C.C. Gregorio, H. Granzier, S. Labeit, The complete gene sequence of titin, expression of an unusual approximately 700-kDa titin isoform, and its interaction with obscurin identify a novel Z-
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[8] C.M. Warren, P.R. Krzesinski, M.L. Greaser, Vertical agarose gel electrophoresis and
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electroblotting of high-molecular-weight proteins, Electrophoresis, 24 (2003) 1695-1702. [9] B.T. Kurien, R.H. Scofield, Ultrarapid electrophoretic transfer of high and low molecular weight proteins using heat, Methods Mol Biol, 536 (2009) 181-190.
[10] W. Gibson, Protease-facilitated transfer of high-molecular-weight proteins during
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electrotransfer to nitrocellulose, Analytical biochemistry, 118 (1981) 1-3. [11] J. Renart, J. Reiser, G.R. Stark, Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure, Proc
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[12] M.W. Bolt, P.A. Mahoney, High-efficiency blotting of proteins of diverse sizes following
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sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Analytical biochemistry, 247 (1997) 185-192.
[13] A. Sumioka, Y. Saito, M. Sakuma, Y. Araki, T. Yamamoto, T. Suzuki, The X11L/X11beta/MINT2 and X11L2/X11gamma/MINT3 scaffold proteins shuttle between the nucleus and cytoplasm, Experimental cell research, 314 (2008) 1155-1162.
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[14] J. O'Sullivan, E. Comerford, W. Rachidi, M. Scott, N.M. Hooper, H.E. McMahon, The effects of the cellular and infectious prion protein on the neuronal adaptor protein X11alpha, Biochim Biophys Acta., 1850 (2015) 2213-2221. doi: 2210.1016/j.bbagen.2015.2208.2010.
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[16] M. Okamoto, T. Matsuyama, M. Sugita, Ultrastructural localization of mint1 at synapses in
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[17] H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc Natl Acad Sci U S A, 76 (1979) 4350-4354.
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Comparison of electroblotting recoveries using different types of PVDF membranes, Analytical biochemistry, 207 (1992) 11-18.
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[21] P.F. Erickson, L.N. Minier, R.S. Lasher, Quantitative electrophoretic transfer of polypeptides from SDS polyacrylamide gels to nitrocellulose sheets: a method for their re-use in immunoautoradiographic detection of antigens, J Immunol Methods, 51 (1982) 241-249.
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Figure legends Figure 1. Diagram of apparatus used for heat transfer method Diagram showing set up of heat
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method including waterbath, mechanical mixing and circulation of water in waterbath via pump.
Figure 2. Comparison of heat western transfer to standard western transfer with modified buffers 20µg of cell line lysates of N2a and N2a22L20Cr cells were analysed by 8% SDS-PAGE
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followed by immunoblotting with X11α antibody. Standard western conditions (1h at 400mA, Room temperature) were used for A-C with a Tris-Glycine buffer (25mM Tris, 192mM Glycine)
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containing the level of methanol (MeOH) and SDS indicated. (D) Western was performed in a TrisGlycine buffer (25mM Tris, 192mM Glycine) without MeOH or SDS. Transfer was carried out at 78C for 1.5h at 400mA. Lanes 1 of A contains 3µg Rat Cerebrum Lysate (BD Biosciences). Protein size is indicated on the left of panels in kDa. Results are representative of three individual
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experiments. Arrow indicates location of full length X11α.
Figure 3. Immobilisation of molecular weight markers using standard and heat western transfer 6µl of commercial pre-strained molecular weight markers were analysed by 8% SDS-
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PAGE (Lanes 1 and 2). Lane 1 sample was transferred onto nitrocellulose by Heat transfer (HT) in
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25mM Tris, 192mM Glycine for 1.5h at 400mA. Lane 2 sample was transferred by standard transfer (1h at 400Ma at RT) with a standard Tris-Glycine buffer (25mM Tris, 192mM Glycine, 20% MeOH, 0.1% SDS). Membranes were imaged simultaneously using white light trans-luminescence. Protein size is indicated on the left of panels in kDa, what these proteins are is indicated on the right. Where more than one entity facilitates the protein size this is a fusion of proteins. Results are representative of three individual experiments.
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Figure 1
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0 0
2
3
4
5
6
7
8
9
N2a
0 0
N2a 22L20Cr
10 0.1
N2a
20 0.1
N2a 22L20Cr
D
N2a
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N2a 22L20Cr
1
B
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Lane 175.0 -
A
N2a
MeOH (%) SDS (%)
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Figure 2
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_____________________________________
Transfer conditions
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N2a 22L20Cr
Rat lysate
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Room Temperature 1h 400mA
_______ 78C 1.5h 400mA
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Figure 3
Lane
1
2
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175.0 -
Maltose binding protein (MBP)-β-galactosidase
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MBP-paramyosin
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MBP-Chitin binding domain (CBD)
46.0 -
CBD-Mxe Intenin-2CBD
30.0 -
CBD-Mxe Intenin
25.0 -
CBD-BmFKBP13
17.0 -
Lysozyme HT
ST
