Chapter 30 Method for Resolution and Western Blotting of Very Large Proteins Using Agarose Electrophoresis Marion L. Greaser and Chad M. Warren Abstract Proteins larger than 200 kDa are difficult to separate electrophoretically using polyacrylamide gels, and their transfer during western blotting is typically incomplete. A vertical SDS agarose gel system was developed that has vastly improved resolving power for very large proteins. Complete transfer of proteins as large as titin (Mr 3,000–3,700 kDa) onto blots can be achieved. The addition of a sulfhydryl reducing agent in the upper reservoir buffer and transfer buffer markedly improves the blotting of large proteins. Key words SeaKem agarose, Titin, DATD, Large protein blotting
1
Introduction Proteins with large subunit size (~>200 kDa) are difficult to separate by electrophoresis because of their poor penetration into gels with the widely used Laemmli SDS (sodium dodecyl sulfate) polyacrylamide system [1]. Protein migration in SDS gels has been found to be linear with the log of the molecular weight [2], so the larger the protein, the more poorly it is resolved from other big proteins. Others have attempted to solve this problem by using very low concentration acrylamide gels [3], acrylamide mixed with agarose [4], or acrylamide gradients [5] to better separate large proteins. Low concentration acrylamide gels are mechanically fragile and distort easily during handling; these problems become magnified when blotting is attempted. An additional difficulty in blotting very large proteins is their poor transfer to the membrane. Inclusion of 2-mercaptoethanol in the transfer buffer improves transfer efficiency, but acrylamide gels stained after transfer typically still contain most of the giant muscle protein titin [6]. An electrophoresis system using SDS and agarose for protein electrophoresis and blotting has been described [7]. An example showing the resolution for several muscle samples containing large
Biji T. Kurien and R. Hal Scofield (eds.), Western Blotting: Methods and Protocols, Methods in Molecular Biology, vol. 1312, DOI 10.1007/978-1-4939-2694-7_30, © Springer Science+Business Media New York 2015
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Fig. 1 SDS 1 % agarose gel stained with silver. A centimeter ruler is shown on the left and the sizes of the various protein bands in kDa are listed on the right. DV dog ventricle, RS rat soleus, HV human ventricle, HS human soleus, CF crayfish claw muscle. Human soleus titin is 3,700 kDa and human ventricle has two titin bands of 3,300 and 3,000 kDa. The bands at 780 and 850 kDa are rat and human nebulin, respectively. The myosin heavy chain is 223 kDa. Blotting proteins this size from acrylamide gels usually results in incomplete transfer, but full transfer can be achieved with agarose [7]
proteins is shown in Fig. 1. Migration distance shows a linear relationship with the log of the molecular weight [7]. This system allows reproducible and quantitative transfer of proteins from the gel in contrast to methods using low percentage acrylamide. The method has been widely adapted for studying muscle proteins, but it also has been used to study protein aggregates of the von Willenbrand Factor [8, 9] and Huntingtin [10].
Agarose Electrophoresis and Blotting
2 2.1
287
Materials Apparatus
1. SE 600 Slab Gel Unit with 16 × 18 cm glass plates (Hoefer) or a similar commercial gel unit (see Note 1). 2. 65 °C Oven. 3. A constant current power supply. 4. Circulating cooler. 5. TE62 Tank Blotting Unit, Hoefer.
2.2
Stock Solutions
1. Acrylamide gel for plug: 38.5 % acrylamide. Weigh 37.5 g of acrylamide and 1 g DATD (N,N′-diallyl-tartardiamide) into a beaker, add about 50 mL of water, stir till dissolved, dilute to 100 mL. Filter through a 0.45 μm filter. The solution should be stored in a brown bottle in the cold room (4 °C). Danger! Avoid skin contact. 2. Reservoir and agarose gel buffer concentrate (5×): 0.25 M Trizma base—1.92 M glycine—0.5 % SDS. Buffer concentrate can be stored at room temperature. 3. Ammonium persulfate; Prepare a 100 mg/mL solution in water; store frozen in 0.5 mL aliquots (stable indefinitely at −20 °C). 4. Sample buffer: 8 M urea, 2 M thiourea, 0.05 M Tris–HCl (pH 6.8), 75 mM DTT, 3 % SDS, 0.05 % bromophenol blue (adapted from ref. 11). (Dissolve urea and thiourea and treat with mixed bed resin to remove ionic constituents; then add remaining ingredients. Store at −20 °C). 5. 50 % v/v glycerol. 6. Transfer buffer: 20 mM Trizma base, 150 mM glycine, 20 % v/v methanol [7, 11] or 10 mM CAPS (N-cyclohexyl-3aminopropanesulfonic acid), pH 11 [12]. For high molecular weight proteins, add SDS and 2-mercaptoethanol to 0.1 % and 10 mM, respectively to the transfer buffer.
3 3.1
Methods Gel Preparation
1. Volumes listed will provide enough solution for two 16 × 18 cm gels with 1.5 mm spacers. One is used for staining either with Coomassie blue or with silver with a special procedure for agarose [7], the other for blotting. 2. Clean plates and spacers with soap, rinse with distilled water and finally with ethanol. 3. Assemble gel plates. Place plate on clean bench top. Place spacers hanging half the way off each side of plate. Place second
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plate on top. Stand up plates and place one side into the clamp. Align spacer with side of plates and clamp and push spacer down so that bottom is flush with the glass plates (top buffer will leak if spacers are not flush with plates). 4. Pour acrylamide plugs in bottom of gel plate assembly (see Note 2): In a 15 mL plastic beaker add: 1.924 mL deionized water, 1.7 mL 50 % glycerol, 2.12 mL 3 M Tris (pH 9.3), 2.72 mL acrylamide (40 %), 24 μL 10 % ammonium persulfate, and 13 μL TEMED (tetramethylethylenediamine) (see Note 2). Mix by pipeting a few times. Immediately add 2.5 mL to each gel assembly. Add a small amount of water on top of each plug to level the upper surface and provide an oxygen barrier. Allow gel to polymerize for 20–30 min. Drain off water layer by inverting gel plate assembly on a paper towel. 5. Place assembly, 20 lane sample combs, and 60 mL plastic syringe in a 65 °C oven for 10 min (see Note 3). 6. Weigh 0.8 g of SeaKem Gold agarose SeaKem Gold Agarose (Lonza Group Ltd) (see Note 4) into a 600 mL beaker (see Note 5). To a 100 mL graduated cylinder add 48 mL of 50 % v/v glycerol (see Note 6), 16 mL 5× electrophoresis buffer, and bring volume up to 80 mL with deionized water. Place Parafilm over top of the graduated cylinder, mix by inverting a few times, and pour solution into the 600 mL beaker containing the agarose. Place Saran wrap over top of beaker and poke a few holes in the Saran wrap. Weigh beaker with contents. Place beaker in a microwave oven along with a separate beaker of deionized water. Heat for a total of 2 min (stop every 30 s to swirl—protect hand with an insulated glove) (see Note 7). 7. Allow agarose to cool for a few minutes at room temperature. Re-weigh, and add sufficient heated deionized water to replace that lost by evaporation. 8. Draw up about 40 mL of agarose in the pre-warmed 60 mL Luer-Lock syringe and pour each gel slowly until it just overflows the top of the plates. Try to avoid formation of bubbles (if bubbles present, bring them to the top of the gel and pinch them with the sample comb). Insert sample combs and allow unit to cool at room temperature for about 45 min (see Note 8). 3.2 Electrophoresis Setup and Sample Loading
1. Add 4 L of buffer to lower chamber (3,200 mL deionized water plus 800 mL 5× electrophoresis buffer). Start cooling unit and stir bar (gels run at 6 °C). 2. Prepare 600 mL upper chamber electrophoresis buffer (same concentration as lower chamber buffer). Add 2-mercaptoethanol (final concentration of 10 mM). Buffer will be poured into top chamber after samples are loaded and assembly placed in unit.
Agarose Electrophoresis and Blotting
289
3. Take combs out of gels by bending them back and forth to detach from gel and slowly pull them up. Pour a small amount of upper chamber buffer into a 15 mL beaker and pipette buffer into first and last wells (the rest will fill over). Add buffer to remove any trapped bubbles. Insert pipette tip to deposit sample in bottom of the sample well. Skip the first and last lanes (see Note 9). 4. Running gels. Once samples are loaded, put upper chamber on the assembly. Pour upper chamber buffer into upper chamber from corners (don’t pour buffer directly over wells). Place lid on unit, and connect to power supply. Turn electrophoresis unit on and run at 30 mA (2 gels) for 3 h. 3.3 Staining and Western Blotting
1. After tracking dye reaches the bottom of the acrylamide plug, turn off the power and disassemble the plates. Cut off sample wells and acrylamide plug and discard. Soak the remaining agarose gel in 10 mM CAPS (pH 11.0), 0.1 % SDS, and 10 mM 2-mercaptoethanol for 30 min with gentle shaking. 2. The gel is then placed on top of either a sheet of PVDF (polyvinylidene difluoride) or nitrocellulose, assembled into the transfer unit, and the protein electrophoretically transferred using 40 V constant voltage for 2–3 h (see Note 10). 3. Blotted proteins can then be treated using conventional procedures with either colorimetric (horseradish peroxidase or alkaline phosphatase substrates) or ECL (enhanced chemiluminescence) methods.
4
Notes 1. The agarose gel procedure works equally well with small format gels (i.e., 8 × 10 cm). 2. The acrylamide plug is used to prevent the agarose from slipping out of the vertical gel plate assembly. Use of DATD as the cross-linker provides a stickier bond of the acrylamide to the glass plates than if a conventional bisacrylamide cross-linker is used. Plugs can be poured a day before making the gel (place tape or Parafilm over the top of the plates to prevent drying and store in cold room). 3. Preheating the glass plate assembly, well comb, and syringe prevents premature agarose gelling when the solution touches the colder surfaces. In addition the plates are less likely to crack during pouring if they are closer to the temperature of the hot agarose. 4. The supplier for SeaKem Gold agarose has changed twice since 2003. Biowhittaker was succeeded by Cambrex who was
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followed by Lonza Group Ltd, Muenchensteinerstrasse 38, CH-4002 Basel, Switzerland. 5. It is essential to use SeaKem Gold agarose for optimal migration of high molecular weight proteins. This type has large pore size and excellent mechanical stability. Other types of agarose may be used, but the protein mobility will be significantly reduced. 6. Glycerol is included in the mixture to increase the solution viscosity inside the gel and thus sharpen the protein bands. 7. Periodic swirling during the heating step eliminates nonhydrated agarose granules in the final gel. 8. Sample combs should extend no longer than 1 cm into agarose; otherwise they may be difficult to remove. Gels can be used right away or stored overnight in a cold room. 9. Conventional sample buffers may not be dense enough for the sample to stay at the bottom of the well. If necessary add additional glycerol (up to 30 % v/v final concentration) to increase sample density. 10. The disulfide bond formation of large proteins during electrophoresis also retards their migration out of the gel onto blots during transfer. Thus inclusion of 2-mercaptoethanol in the transfer buffer improves efficiency of transfer of high molecular weight proteins. The use of the agarose electrophoresis system with inclusion of 2-mercaptoethanol in the transfer buffer results in complete transfer of all high molecular weight proteins out of the gel, including titin (Mr 3,000–3,700 kDa subunit size) [7]. Alternatively, protein can be alkylated to prevent disulfide bond formation during the transfer process [13].
Acknowledgements This work was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison and from grants (MLG-NIH HL77196 and Hatch NC1184). References 1. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 2. Weber K, Osborn M (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412 3. Granzier H, Wang K (1993) Gel electrophoresis of giant proteins: solubilization and silver-
staining of titin and nebulin from single muscle fiber segments. Electrophoresis 14:56–64 4. Tatsumi R, Hattori A (1995) Detection of giant myofibrillar proteins connectin and nebulin by electrophoresis in 2 % polyacrylamide slab gels strengthened with agarose. Anal Biochem 224:28–31 5. Cazorla O, Freiburg A, Helmes M, Centner T, McNabb M, Wu Y, Trombitas K, Labeit S,
Agarose Electrophoresis and Blotting
6.
7.
8.
9.
Granzier H (2000) Differential expression of cardiac titin isoforms and modulation of cellular stiffness. Circ Res 86:59–67 Fritz JD, Swartz DR, Greaser ML (1989) Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins. Anal Biochem 180:205–210 Warren CM, Krzesinski PR, Greaser ML (2003) Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins. Electrophoresis 24:1695–1702 Ott HW, Griesmache A, Schnapka-Koepf M, Golderer G, Sieberer A, Spannagl M, Scheibe B, Perkhofer S, Will K, Budde U (2010) Analysis of von Willebrand Factor multimers by simultaneous high- and low-resolution vertical SDS-agarose gel electrophoresis and Cy5labeled antibody high-sensitivity fluorescence detection. Am J Clin Pathol 133:322–330 Akiyama R, Komori I, Hiramoto R, Isonishi A, Matsumoto M, Fujimura Y (2011) H1N1
10.
11.
12.
13.
291
influenza (swine flu)-associated thrombotic microangiopathy with a markedly high plasma Ratio of von Willebrand Factor to ADAMTS13. Intern Med 50:643–647 Hoffner G, Island M-L, Djian P (2005) Purification of neuronal inclusions of patients with Huntington’s disease reveals a broad range of N-terminal fragments of expanded huntingtin and insoluble polymers. J Neurochem 95:125–136 Yates LD, Greaser ML (1983) Quantitative determination of myosin and actin in rabbit skeletal muscle. J Mol Biol 168:123–141 Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262:10035–10038 Sechi S, Chait BT (1998) Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification. Anal Chem 70:5150–5158
1
Introduction Proteins with large subunit size (~>200 kDa) are difficult to separate by electrophoresis because of their poor penetration into gels with the widely used Laemmli SDS (sodium dodecyl sulfate) polyacrylamide system [1]. Protein migration in SDS gels has been found to be linear with the log of the molecular weight [2], so the larger the protein, the more poorly it is resolved from other big proteins. Others have attempted to solve this problem by using very low concentration acrylamide gels [3], acrylamide mixed with agarose [4], or acrylamide gradients [5] to better separate large proteins. Low concentration acrylamide gels are mechanically fragile and distort easily during handling; these problems become magnified when blotting is attempted. An additional difficulty in blotting very large proteins is their poor transfer to the membrane. Inclusion of 2-mercaptoethanol in the transfer buffer improves transfer efficiency, but acrylamide gels stained after transfer typically still contain most of the giant muscle protein titin [6]. An electrophoresis system using SDS and agarose for protein electrophoresis and blotting has been described [7]. An example showing the resolution for several muscle samples containing large
Biji T. Kurien and R. Hal Scofield (eds.), Western Blotting: Methods and Protocols, Methods in Molecular Biology, vol. 1312, DOI 10.1007/978-1-4939-2694-7_30, © Springer Science+Business Media New York 2015
285
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Marion L. Greaser and Chad M. Warren
Fig. 1 SDS 1 % agarose gel stained with silver. A centimeter ruler is shown on the left and the sizes of the various protein bands in kDa are listed on the right. DV dog ventricle, RS rat soleus, HV human ventricle, HS human soleus, CF crayfish claw muscle. Human soleus titin is 3,700 kDa and human ventricle has two titin bands of 3,300 and 3,000 kDa. The bands at 780 and 850 kDa are rat and human nebulin, respectively. The myosin heavy chain is 223 kDa. Blotting proteins this size from acrylamide gels usually results in incomplete transfer, but full transfer can be achieved with agarose [7]
proteins is shown in Fig. 1. Migration distance shows a linear relationship with the log of the molecular weight [7]. This system allows reproducible and quantitative transfer of proteins from the gel in contrast to methods using low percentage acrylamide. The method has been widely adapted for studying muscle proteins, but it also has been used to study protein aggregates of the von Willenbrand Factor [8, 9] and Huntingtin [10].
Agarose Electrophoresis and Blotting
2 2.1
287
Materials Apparatus
1. SE 600 Slab Gel Unit with 16 × 18 cm glass plates (Hoefer) or a similar commercial gel unit (see Note 1). 2. 65 °C Oven. 3. A constant current power supply. 4. Circulating cooler. 5. TE62 Tank Blotting Unit, Hoefer.
2.2
Stock Solutions
1. Acrylamide gel for plug: 38.5 % acrylamide. Weigh 37.5 g of acrylamide and 1 g DATD (N,N′-diallyl-tartardiamide) into a beaker, add about 50 mL of water, stir till dissolved, dilute to 100 mL. Filter through a 0.45 μm filter. The solution should be stored in a brown bottle in the cold room (4 °C). Danger! Avoid skin contact. 2. Reservoir and agarose gel buffer concentrate (5×): 0.25 M Trizma base—1.92 M glycine—0.5 % SDS. Buffer concentrate can be stored at room temperature. 3. Ammonium persulfate; Prepare a 100 mg/mL solution in water; store frozen in 0.5 mL aliquots (stable indefinitely at −20 °C). 4. Sample buffer: 8 M urea, 2 M thiourea, 0.05 M Tris–HCl (pH 6.8), 75 mM DTT, 3 % SDS, 0.05 % bromophenol blue (adapted from ref. 11). (Dissolve urea and thiourea and treat with mixed bed resin to remove ionic constituents; then add remaining ingredients. Store at −20 °C). 5. 50 % v/v glycerol. 6. Transfer buffer: 20 mM Trizma base, 150 mM glycine, 20 % v/v methanol [7, 11] or 10 mM CAPS (N-cyclohexyl-3aminopropanesulfonic acid), pH 11 [12]. For high molecular weight proteins, add SDS and 2-mercaptoethanol to 0.1 % and 10 mM, respectively to the transfer buffer.
3 3.1
Methods Gel Preparation
1. Volumes listed will provide enough solution for two 16 × 18 cm gels with 1.5 mm spacers. One is used for staining either with Coomassie blue or with silver with a special procedure for agarose [7], the other for blotting. 2. Clean plates and spacers with soap, rinse with distilled water and finally with ethanol. 3. Assemble gel plates. Place plate on clean bench top. Place spacers hanging half the way off each side of plate. Place second
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Marion L. Greaser and Chad M. Warren
plate on top. Stand up plates and place one side into the clamp. Align spacer with side of plates and clamp and push spacer down so that bottom is flush with the glass plates (top buffer will leak if spacers are not flush with plates). 4. Pour acrylamide plugs in bottom of gel plate assembly (see Note 2): In a 15 mL plastic beaker add: 1.924 mL deionized water, 1.7 mL 50 % glycerol, 2.12 mL 3 M Tris (pH 9.3), 2.72 mL acrylamide (40 %), 24 μL 10 % ammonium persulfate, and 13 μL TEMED (tetramethylethylenediamine) (see Note 2). Mix by pipeting a few times. Immediately add 2.5 mL to each gel assembly. Add a small amount of water on top of each plug to level the upper surface and provide an oxygen barrier. Allow gel to polymerize for 20–30 min. Drain off water layer by inverting gel plate assembly on a paper towel. 5. Place assembly, 20 lane sample combs, and 60 mL plastic syringe in a 65 °C oven for 10 min (see Note 3). 6. Weigh 0.8 g of SeaKem Gold agarose SeaKem Gold Agarose (Lonza Group Ltd) (see Note 4) into a 600 mL beaker (see Note 5). To a 100 mL graduated cylinder add 48 mL of 50 % v/v glycerol (see Note 6), 16 mL 5× electrophoresis buffer, and bring volume up to 80 mL with deionized water. Place Parafilm over top of the graduated cylinder, mix by inverting a few times, and pour solution into the 600 mL beaker containing the agarose. Place Saran wrap over top of beaker and poke a few holes in the Saran wrap. Weigh beaker with contents. Place beaker in a microwave oven along with a separate beaker of deionized water. Heat for a total of 2 min (stop every 30 s to swirl—protect hand with an insulated glove) (see Note 7). 7. Allow agarose to cool for a few minutes at room temperature. Re-weigh, and add sufficient heated deionized water to replace that lost by evaporation. 8. Draw up about 40 mL of agarose in the pre-warmed 60 mL Luer-Lock syringe and pour each gel slowly until it just overflows the top of the plates. Try to avoid formation of bubbles (if bubbles present, bring them to the top of the gel and pinch them with the sample comb). Insert sample combs and allow unit to cool at room temperature for about 45 min (see Note 8). 3.2 Electrophoresis Setup and Sample Loading
1. Add 4 L of buffer to lower chamber (3,200 mL deionized water plus 800 mL 5× electrophoresis buffer). Start cooling unit and stir bar (gels run at 6 °C). 2. Prepare 600 mL upper chamber electrophoresis buffer (same concentration as lower chamber buffer). Add 2-mercaptoethanol (final concentration of 10 mM). Buffer will be poured into top chamber after samples are loaded and assembly placed in unit.
Agarose Electrophoresis and Blotting
289
3. Take combs out of gels by bending them back and forth to detach from gel and slowly pull them up. Pour a small amount of upper chamber buffer into a 15 mL beaker and pipette buffer into first and last wells (the rest will fill over). Add buffer to remove any trapped bubbles. Insert pipette tip to deposit sample in bottom of the sample well. Skip the first and last lanes (see Note 9). 4. Running gels. Once samples are loaded, put upper chamber on the assembly. Pour upper chamber buffer into upper chamber from corners (don’t pour buffer directly over wells). Place lid on unit, and connect to power supply. Turn electrophoresis unit on and run at 30 mA (2 gels) for 3 h. 3.3 Staining and Western Blotting
1. After tracking dye reaches the bottom of the acrylamide plug, turn off the power and disassemble the plates. Cut off sample wells and acrylamide plug and discard. Soak the remaining agarose gel in 10 mM CAPS (pH 11.0), 0.1 % SDS, and 10 mM 2-mercaptoethanol for 30 min with gentle shaking. 2. The gel is then placed on top of either a sheet of PVDF (polyvinylidene difluoride) or nitrocellulose, assembled into the transfer unit, and the protein electrophoretically transferred using 40 V constant voltage for 2–3 h (see Note 10). 3. Blotted proteins can then be treated using conventional procedures with either colorimetric (horseradish peroxidase or alkaline phosphatase substrates) or ECL (enhanced chemiluminescence) methods.
4
Notes 1. The agarose gel procedure works equally well with small format gels (i.e., 8 × 10 cm). 2. The acrylamide plug is used to prevent the agarose from slipping out of the vertical gel plate assembly. Use of DATD as the cross-linker provides a stickier bond of the acrylamide to the glass plates than if a conventional bisacrylamide cross-linker is used. Plugs can be poured a day before making the gel (place tape or Parafilm over the top of the plates to prevent drying and store in cold room). 3. Preheating the glass plate assembly, well comb, and syringe prevents premature agarose gelling when the solution touches the colder surfaces. In addition the plates are less likely to crack during pouring if they are closer to the temperature of the hot agarose. 4. The supplier for SeaKem Gold agarose has changed twice since 2003. Biowhittaker was succeeded by Cambrex who was
290
Marion L. Greaser and Chad M. Warren
followed by Lonza Group Ltd, Muenchensteinerstrasse 38, CH-4002 Basel, Switzerland. 5. It is essential to use SeaKem Gold agarose for optimal migration of high molecular weight proteins. This type has large pore size and excellent mechanical stability. Other types of agarose may be used, but the protein mobility will be significantly reduced. 6. Glycerol is included in the mixture to increase the solution viscosity inside the gel and thus sharpen the protein bands. 7. Periodic swirling during the heating step eliminates nonhydrated agarose granules in the final gel. 8. Sample combs should extend no longer than 1 cm into agarose; otherwise they may be difficult to remove. Gels can be used right away or stored overnight in a cold room. 9. Conventional sample buffers may not be dense enough for the sample to stay at the bottom of the well. If necessary add additional glycerol (up to 30 % v/v final concentration) to increase sample density. 10. The disulfide bond formation of large proteins during electrophoresis also retards their migration out of the gel onto blots during transfer. Thus inclusion of 2-mercaptoethanol in the transfer buffer improves efficiency of transfer of high molecular weight proteins. The use of the agarose electrophoresis system with inclusion of 2-mercaptoethanol in the transfer buffer results in complete transfer of all high molecular weight proteins out of the gel, including titin (Mr 3,000–3,700 kDa subunit size) [7]. Alternatively, protein can be alkylated to prevent disulfide bond formation during the transfer process [13].
Acknowledgements This work was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison and from grants (MLG-NIH HL77196 and Hatch NC1184). References 1. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 2. Weber K, Osborn M (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412 3. Granzier H, Wang K (1993) Gel electrophoresis of giant proteins: solubilization and silver-
staining of titin and nebulin from single muscle fiber segments. Electrophoresis 14:56–64 4. Tatsumi R, Hattori A (1995) Detection of giant myofibrillar proteins connectin and nebulin by electrophoresis in 2 % polyacrylamide slab gels strengthened with agarose. Anal Biochem 224:28–31 5. Cazorla O, Freiburg A, Helmes M, Centner T, McNabb M, Wu Y, Trombitas K, Labeit S,
Agarose Electrophoresis and Blotting
6.
7.
8.
9.
Granzier H (2000) Differential expression of cardiac titin isoforms and modulation of cellular stiffness. Circ Res 86:59–67 Fritz JD, Swartz DR, Greaser ML (1989) Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins. Anal Biochem 180:205–210 Warren CM, Krzesinski PR, Greaser ML (2003) Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins. Electrophoresis 24:1695–1702 Ott HW, Griesmache A, Schnapka-Koepf M, Golderer G, Sieberer A, Spannagl M, Scheibe B, Perkhofer S, Will K, Budde U (2010) Analysis of von Willebrand Factor multimers by simultaneous high- and low-resolution vertical SDS-agarose gel electrophoresis and Cy5labeled antibody high-sensitivity fluorescence detection. Am J Clin Pathol 133:322–330 Akiyama R, Komori I, Hiramoto R, Isonishi A, Matsumoto M, Fujimura Y (2011) H1N1
10.
11.
12.
13.
291
influenza (swine flu)-associated thrombotic microangiopathy with a markedly high plasma Ratio of von Willebrand Factor to ADAMTS13. Intern Med 50:643–647 Hoffner G, Island M-L, Djian P (2005) Purification of neuronal inclusions of patients with Huntington’s disease reveals a broad range of N-terminal fragments of expanded huntingtin and insoluble polymers. J Neurochem 95:125–136 Yates LD, Greaser ML (1983) Quantitative determination of myosin and actin in rabbit skeletal muscle. J Mol Biol 168:123–141 Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262:10035–10038 Sechi S, Chait BT (1998) Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification. Anal Chem 70:5150–5158
