Chapter 16 Protein Blotting Protocol for Beginners Lars A. Petrasovits Abstract The transfer and immobilization of biological macromolecules onto solid nitrocellulose or nylon (polyvinylidene difluoride (PVDF)) membranes subsequently followed by specific detection is referred to as blotting. DNA blots are called Southerns after the inventor of the technique, Edwin Southern. By analogy, RNA blots are referred to as northerns and protein blots as westerns [1]. With few exceptions, western blotting involves five steps, namely, sample collection, preparation, separation, immobilization, and detection. In this chapter, protocols for the entire process from sample collection to detection are described. Key words Protein extraction, Trizol, SDS-PAGE, Electrophoretic transfer, Immunodetection
1
Introduction
1.1 Sample Collection and Preparation
Proteins are subject to a plethora of posttranscriptional and posttranslational chemical modifications and therefore, speed is of the essence when samples are harvested. The most common procedures involve snap-freezing in a liquid nitrogen or ethanol–dry ice bath. Alternatively, immediate sample processing may be employed in order to minimize the introduction of experimental error. The first step is usually disruption of solid samples. This is achieved mechanically by blending, grinding, sonification, or enzymatic digestion in a suitable lysis buffer depending on the purpose of the analysis. Lysis buffers can vary in their composition but usually contain a buffer such as MOPS, HEPES, or Tris-buffered saline (TBS), metal-chelates such as EDTA or EGTA, detergents (denaturants) such as SDS, Triton X-100, Nonidet-P40 or deoxycholate, and reducing agents such as β-mercaptoethanol or dithiothreitol. Additionally, protease inhibitors or chaotropic agents may be present to aid in cell disruption. There are several lysis buffer preparations commercially available, such as Invitrogen’s Trizol® and Sigma’s TRI Reagent® that allow for multiple macromolecular species to be extracted from the same sample [2]. The next step is
Robert J. Henry and Agnelo Furtado (eds.), Cereal Genomics: Methods and Protocols, Methods in Molecular Biology, vol. 1099, DOI 10.1007/978-1-62703-715-0_16, © Springer Science+Business Media New York 2014
189
190
Lars A. Petrasovits
Table 1 Gel strength and approximate separation capacity Gel percentage (%)
Size range (kDA)
8
25–200
10
15–100
12.5
10–70
15
12–45
20
4–40
the removal of cellular debris and other contaminants from the sample. This is usually achieved by filtration, centrifugation, dialysis, and/or precipitation of proteins. After this, a buffer compatible with the separation system (Sample loading buffer) is added to the sample. It is often desirable to carry out sample preparation at low temperatures and minimize sample handling to avoid artifacts. Prepared samples can be stored at below −20 °C. 1.2
Separation
To most common method of separation of proteins is polyacrylamide gel electrophoresis (PAGE) [3]. Separation is achieved based on size and charge of a given protein. In contrast to nucleic acids that have a net negative charge per nucleotide base, proteins are not charge-uniform. This is important when native (without reductants or detergents) PAGE of highly basic proteins such as histones is attempted. The inclusion of SDS serves two purposes: disruption of secondary (α-helices and β-pleated sheets), tertiary (electrostatic, but not disulfide bonds that are reduced), and quaternary (three-dimensional monomer assembly) protein structure and coating of the primary structure to provide a net negative charge, so that proteins will move to the positively charged electrode. In SDS-PAGE, proteins migrate through the gel at a rate inversely proportional to their size. The pore size of gels determines the size range of proteins that can be separated and is dependent on acrylamide plus cross-linker (N,Nmethylenebisacrylamide) concentration (Table 1). Since acrylamide is a powerful cumulative neurotoxin, it is NOT recommended that this should be attempted by a complete novice! Readymade gels can be purchased, for example Nu-Page™ from Invitrogen or Mini-Protean™ from Bio-Rad. Commercially supplied gels are guaranteed to perform consistently, both within the same batch or between batches and are cheaper to use than homemade gels. Both types have been successfully used in our laboratory; however, gels and electrophoresis equipment are not compatible or interchangeable due to differences in buffer systems and physical size of the gels.
Protein Blotting
191
Fig. 1 A polyacrylamide gel showing proteins resolved by electrophoresis and then stained
Pre-electrophoretic steps are the setting up of the gel cells and sample loading: readymade gels are removed from packaging, combs and tape removed, and the wells thoroughly rinsed with water followed by running buffer (typically 25 mM Tris, 192 mM glycine, pH 8.3 plus 0.1 % SDS for SDS-PAGE). The sample may be boiled for 5 min before loading. Molecular weight standards and a known positive and negative control should be included. It is important to read the manufacturer’s instructions to determine the gel capacity, typically around 10 μg of protein per well. Sample concentrations may be determined using Lowry or Bradford assays or can be done by measuring A280. Each method has intrinsic flaws, so that perhaps the best method to ensure concentration uniformity across multiple samples is to run them on a gel, staining with Coomassie Blue or similar and adjusting concentrations based on the intensity of multiple protein bands (Fig. 1). For most gels, run times are around 30 min at 100–200 V or until the loading has migrated to the bottom of the gel. The gel is then removed from its cast and may be rinsed in blotting buffer for several minutes. 1.3 Transfer and Immobilization
Transfer of proteins from the gel onto the membrane is carried out in an electric field and follows the same principle as electrophoresis. SDS-coated proteins are negatively charged and migrate towards the anode accordingly. The difference is that membrane and gel are in direct contact and the membrane stops proteins from further travel. Gel and membrane are sandwiched between filter paper and sponges (sponge–paper–gel–membrane– paper–sponge) and submerged in transfer buffer so that the gel faces the cathode. There are several different transfer buffers to choose from and some consideration needs to be given as to their
192
Lars A. Petrasovits
suitability: High pH buffers (e.g. 10 mM NaHCO3, 3 mM Na2CO3, 20 % methanol, pH 9.9) improve binding of proteins to positively charged membranes [4]. Some protocols recommend addition of 20 % methanol but this is not necessary if PVDF membranes are used. Methanol reduces pore size of gels making transfer of large proteins more difficult [5]. In contrast to nitrocellulose, PVDF membranes require pre-wetting with methanol or ethanol followed by equilibration in transfer buffer. Also, presence of SDS in the buffer increases elution efficiency of proteins from gels but reduces their binding efficiency to the membrane, increases ionic strength of the transfer buffer and may precipitate from solution at low temperature [6]. 1.4
2
Detection
When native PAGE is employed as a separation method, detection of a protein of interest may be achieved by activity or cofactor binding assays [7]. However, the majority of proteins are denatured and reduced after being subjected to SDS-PAGE and therefore do not retain biological activity. The most common method of detection is by immunological means involving the use of specific antisera raised against the protein of interest. Since antibodies are proteins, they will bind to the membranes nonspecifically. This is overcome by the use of blocking agents, and the most commonly used ones are bovine serum albumin and skim milk powder. After blocking, the membrane is incubated with the primary antibody specific to the protein of interest, typically a rabbit or mouse immunoglobulin (IgG). Many antisera are now commercially available and are commonly used at a 1:1,000 dilution. After removal of excess/unbound primary antibody, a secondary antibody against the primary antibody conjugated to a reporter system such as immunogold, horseradish peroxidase (HRP), or alkaline phosphatase (AP) is added. Again, unbound antibodies are removed and detection is achieved either through the use of chromogenic or chemiluminescent substrates. Secondary antibody/ detection kits are commercially available, e.g., Invitrogen’s Western Breeze® or Amersham’s ECL Plus™ kits.
Materials
2.1 Sample Collection and Preparation Using Trizol®
1. Liquid Nitrogen. 2. Trizol. 3. Chloroform. 4. Isopropyl alcohol. 5. 75 % Ethanol v/v in DEPC-treated water. 6. RNase-free water or 0.5 % SDS solution; To prepare RNasefree water, draw water into RNase-free glass bottles.
Protein Blotting
193
Add diethylpyrocarbonate (DEPC) to 0.01 % (v/v). Incubate overnight at room temperature and autoclave. The SDS solution must be prepared using DEPC-treated autoclaved water. 7. 100 % Ethanol. 8. 0.1 M Sodium citrate in 10 % ethanol (v/v in distilled water). 9. 0.3 M Guanidine hydrochloride in 95 % ethanol. 10. 1 % SDS or Sample buffer (32 mM Tris–HCl, pH 6.8, 1 % (w/v) SDS, 12.5 % (v/v) glycerol, 0.005 % (w/v) Bromophenol Blue, 2.5 % (v/v) 350 mM DTT (added fresh). 2.2 Separation of Proteins by SDS-PAGE
1. Electrophoresis tank. 2. Electrophoresis module; Bio-Rad Mini-PROTEAN® Tetra cell equipment (cat #s 165-8000 and 165-800, Bio-Rad, USA). 3. Power pack. 4. Precast gels; Mini-PROTEAN® precast gels (Bio-Rad, USA). 5. Buffer dam (if only one gel is run). 6. Running buffer (700 mL: 25 mM Tris-base, 192 mM glycine, 0.1 % (w/v) SDS, pH 8.3). 7. Samples (1× sample buffer: 32 mM Tris–HCl, pH 6.8, 1 % (w/v) SDS, 12.5 % (v/v) glycerol, 0.005 % (w/v) Bromophenol Blue, 2.5 % (v/v) 350 mM DTT (added fresh). 8. Protein size marker. 9. Bio-Safe Coomassie G-250 stain (Bio-Rad, cat.#161-0786).
2.3 Electrophoretic Transfer and Immobilization
1. Electrophoresis tank; Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell (Cat # 170-3930, Bio-Rad, USA). 2. Power pack. 3. Gel, equilibrated in transfer buffer. 4. Transfer buffer (25 mM Tris-base, 192 mM glycine, 0.1 % (w/v) SDS, pH 8.3). 5. PVDF membrane. 6. Gel holder cassette, fiber pads, filter paper, electrode module. 7. Blue cooling unit, frozen. 8. Magnetic stirrer and flea.
2.4 Detection Using Western Breeze®
1. Western Breeze Kit (Invitrogen, cat #s WB7104, WB7106, and WB7106). 2. Blotted membrane. 3. Primary antibody. 4. Sterile distilled water. 5. Clean dishes.
194
Lars A. Petrasovits
6. Forceps for handling membranes. 7. Orbital shaker, set to 1 rev/min. 8. X-ray film, dark room, and film development chemicals. 9. Autoradiography cassette (Kodak X-OMAT AR or equivalent).
3
Method
3.1 Sample Collection and Preparation Using Trizol®
Caution: When working with TRIZOL Reagent use gloves and eye protection (shield, safety goggles). Avoid contact with skin or clothing. Use in a chemical fume hood. Avoid breathing vapor. TRIZOL® (Invitrogen Cat. No. 15596-026) is a registered trademark of Molecular Research Center, Inc. A recipe for Trizol is provided (Table 2). Unless otherwise stated, the procedure is carried out at 15–30 °C, and reagents are at 15–30 °C. 1. Grind Tissue under Liquid Nitrogen with a mortar and pestle until a fine powder is obtained. 2. Using a cold spatula, weigh out up to 100 mg of frozen tissue powder into 10 volumes of Trizol (see Notes 1–3). 3. Incubate for at least 5 min at room temperature (RT), then centrifuge for 5 min at 12,000 × g. Transfer supernatant to a fresh tube. 4. Add 0.2 volumes of chloroform and shake vigorously for 15 s and incubate for 2–3 min (see Note 4). 5. Remove the aqueous phase (ca. 60 % of total volume) containing RNA. The RNA is precipitated with 0.5 volumes
Table 2 Homemade recipe for 1 L of Trizol
Reagents
Volume/mass
Final concentration
Acid Phenol
380 mL
38 %
Guanidine thiocyanate
94.53 g
0.8 M
Ammonium thiocyanate
30.45 g
0.4 M
Sodium acetate, pH 5.0
33.4 mL of 3 M stock
0.1 M
Glycerol
50 mL
5%
RNase-free water
Adjust final volume to 1 L
Source: http://ipmb.sinica.edu.tw/microarray/newprotocol/TRI.pdf
Protein Blotting
195
Isopropanol per volume Trizol, centrifugation at 12,000 × g, 4 °C and stored under 75 % ethanol at ≤20 °C. 6. Add 0.3 volumes ethanol per volume Trizol used for homogenization to the organic phase. Mix samples by inversion, incubate at RT for 3 min and centrifuge at
1
Introduction
1.1 Sample Collection and Preparation
Proteins are subject to a plethora of posttranscriptional and posttranslational chemical modifications and therefore, speed is of the essence when samples are harvested. The most common procedures involve snap-freezing in a liquid nitrogen or ethanol–dry ice bath. Alternatively, immediate sample processing may be employed in order to minimize the introduction of experimental error. The first step is usually disruption of solid samples. This is achieved mechanically by blending, grinding, sonification, or enzymatic digestion in a suitable lysis buffer depending on the purpose of the analysis. Lysis buffers can vary in their composition but usually contain a buffer such as MOPS, HEPES, or Tris-buffered saline (TBS), metal-chelates such as EDTA or EGTA, detergents (denaturants) such as SDS, Triton X-100, Nonidet-P40 or deoxycholate, and reducing agents such as β-mercaptoethanol or dithiothreitol. Additionally, protease inhibitors or chaotropic agents may be present to aid in cell disruption. There are several lysis buffer preparations commercially available, such as Invitrogen’s Trizol® and Sigma’s TRI Reagent® that allow for multiple macromolecular species to be extracted from the same sample [2]. The next step is
Robert J. Henry and Agnelo Furtado (eds.), Cereal Genomics: Methods and Protocols, Methods in Molecular Biology, vol. 1099, DOI 10.1007/978-1-62703-715-0_16, © Springer Science+Business Media New York 2014
189
190
Lars A. Petrasovits
Table 1 Gel strength and approximate separation capacity Gel percentage (%)
Size range (kDA)
8
25–200
10
15–100
12.5
10–70
15
12–45
20
4–40
the removal of cellular debris and other contaminants from the sample. This is usually achieved by filtration, centrifugation, dialysis, and/or precipitation of proteins. After this, a buffer compatible with the separation system (Sample loading buffer) is added to the sample. It is often desirable to carry out sample preparation at low temperatures and minimize sample handling to avoid artifacts. Prepared samples can be stored at below −20 °C. 1.2
Separation
To most common method of separation of proteins is polyacrylamide gel electrophoresis (PAGE) [3]. Separation is achieved based on size and charge of a given protein. In contrast to nucleic acids that have a net negative charge per nucleotide base, proteins are not charge-uniform. This is important when native (without reductants or detergents) PAGE of highly basic proteins such as histones is attempted. The inclusion of SDS serves two purposes: disruption of secondary (α-helices and β-pleated sheets), tertiary (electrostatic, but not disulfide bonds that are reduced), and quaternary (three-dimensional monomer assembly) protein structure and coating of the primary structure to provide a net negative charge, so that proteins will move to the positively charged electrode. In SDS-PAGE, proteins migrate through the gel at a rate inversely proportional to their size. The pore size of gels determines the size range of proteins that can be separated and is dependent on acrylamide plus cross-linker (N,Nmethylenebisacrylamide) concentration (Table 1). Since acrylamide is a powerful cumulative neurotoxin, it is NOT recommended that this should be attempted by a complete novice! Readymade gels can be purchased, for example Nu-Page™ from Invitrogen or Mini-Protean™ from Bio-Rad. Commercially supplied gels are guaranteed to perform consistently, both within the same batch or between batches and are cheaper to use than homemade gels. Both types have been successfully used in our laboratory; however, gels and electrophoresis equipment are not compatible or interchangeable due to differences in buffer systems and physical size of the gels.
Protein Blotting
191
Fig. 1 A polyacrylamide gel showing proteins resolved by electrophoresis and then stained
Pre-electrophoretic steps are the setting up of the gel cells and sample loading: readymade gels are removed from packaging, combs and tape removed, and the wells thoroughly rinsed with water followed by running buffer (typically 25 mM Tris, 192 mM glycine, pH 8.3 plus 0.1 % SDS for SDS-PAGE). The sample may be boiled for 5 min before loading. Molecular weight standards and a known positive and negative control should be included. It is important to read the manufacturer’s instructions to determine the gel capacity, typically around 10 μg of protein per well. Sample concentrations may be determined using Lowry or Bradford assays or can be done by measuring A280. Each method has intrinsic flaws, so that perhaps the best method to ensure concentration uniformity across multiple samples is to run them on a gel, staining with Coomassie Blue or similar and adjusting concentrations based on the intensity of multiple protein bands (Fig. 1). For most gels, run times are around 30 min at 100–200 V or until the loading has migrated to the bottom of the gel. The gel is then removed from its cast and may be rinsed in blotting buffer for several minutes. 1.3 Transfer and Immobilization
Transfer of proteins from the gel onto the membrane is carried out in an electric field and follows the same principle as electrophoresis. SDS-coated proteins are negatively charged and migrate towards the anode accordingly. The difference is that membrane and gel are in direct contact and the membrane stops proteins from further travel. Gel and membrane are sandwiched between filter paper and sponges (sponge–paper–gel–membrane– paper–sponge) and submerged in transfer buffer so that the gel faces the cathode. There are several different transfer buffers to choose from and some consideration needs to be given as to their
192
Lars A. Petrasovits
suitability: High pH buffers (e.g. 10 mM NaHCO3, 3 mM Na2CO3, 20 % methanol, pH 9.9) improve binding of proteins to positively charged membranes [4]. Some protocols recommend addition of 20 % methanol but this is not necessary if PVDF membranes are used. Methanol reduces pore size of gels making transfer of large proteins more difficult [5]. In contrast to nitrocellulose, PVDF membranes require pre-wetting with methanol or ethanol followed by equilibration in transfer buffer. Also, presence of SDS in the buffer increases elution efficiency of proteins from gels but reduces their binding efficiency to the membrane, increases ionic strength of the transfer buffer and may precipitate from solution at low temperature [6]. 1.4
2
Detection
When native PAGE is employed as a separation method, detection of a protein of interest may be achieved by activity or cofactor binding assays [7]. However, the majority of proteins are denatured and reduced after being subjected to SDS-PAGE and therefore do not retain biological activity. The most common method of detection is by immunological means involving the use of specific antisera raised against the protein of interest. Since antibodies are proteins, they will bind to the membranes nonspecifically. This is overcome by the use of blocking agents, and the most commonly used ones are bovine serum albumin and skim milk powder. After blocking, the membrane is incubated with the primary antibody specific to the protein of interest, typically a rabbit or mouse immunoglobulin (IgG). Many antisera are now commercially available and are commonly used at a 1:1,000 dilution. After removal of excess/unbound primary antibody, a secondary antibody against the primary antibody conjugated to a reporter system such as immunogold, horseradish peroxidase (HRP), or alkaline phosphatase (AP) is added. Again, unbound antibodies are removed and detection is achieved either through the use of chromogenic or chemiluminescent substrates. Secondary antibody/ detection kits are commercially available, e.g., Invitrogen’s Western Breeze® or Amersham’s ECL Plus™ kits.
Materials
2.1 Sample Collection and Preparation Using Trizol®
1. Liquid Nitrogen. 2. Trizol. 3. Chloroform. 4. Isopropyl alcohol. 5. 75 % Ethanol v/v in DEPC-treated water. 6. RNase-free water or 0.5 % SDS solution; To prepare RNasefree water, draw water into RNase-free glass bottles.
Protein Blotting
193
Add diethylpyrocarbonate (DEPC) to 0.01 % (v/v). Incubate overnight at room temperature and autoclave. The SDS solution must be prepared using DEPC-treated autoclaved water. 7. 100 % Ethanol. 8. 0.1 M Sodium citrate in 10 % ethanol (v/v in distilled water). 9. 0.3 M Guanidine hydrochloride in 95 % ethanol. 10. 1 % SDS or Sample buffer (32 mM Tris–HCl, pH 6.8, 1 % (w/v) SDS, 12.5 % (v/v) glycerol, 0.005 % (w/v) Bromophenol Blue, 2.5 % (v/v) 350 mM DTT (added fresh). 2.2 Separation of Proteins by SDS-PAGE
1. Electrophoresis tank. 2. Electrophoresis module; Bio-Rad Mini-PROTEAN® Tetra cell equipment (cat #s 165-8000 and 165-800, Bio-Rad, USA). 3. Power pack. 4. Precast gels; Mini-PROTEAN® precast gels (Bio-Rad, USA). 5. Buffer dam (if only one gel is run). 6. Running buffer (700 mL: 25 mM Tris-base, 192 mM glycine, 0.1 % (w/v) SDS, pH 8.3). 7. Samples (1× sample buffer: 32 mM Tris–HCl, pH 6.8, 1 % (w/v) SDS, 12.5 % (v/v) glycerol, 0.005 % (w/v) Bromophenol Blue, 2.5 % (v/v) 350 mM DTT (added fresh). 8. Protein size marker. 9. Bio-Safe Coomassie G-250 stain (Bio-Rad, cat.#161-0786).
2.3 Electrophoretic Transfer and Immobilization
1. Electrophoresis tank; Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell (Cat # 170-3930, Bio-Rad, USA). 2. Power pack. 3. Gel, equilibrated in transfer buffer. 4. Transfer buffer (25 mM Tris-base, 192 mM glycine, 0.1 % (w/v) SDS, pH 8.3). 5. PVDF membrane. 6. Gel holder cassette, fiber pads, filter paper, electrode module. 7. Blue cooling unit, frozen. 8. Magnetic stirrer and flea.
2.4 Detection Using Western Breeze®
1. Western Breeze Kit (Invitrogen, cat #s WB7104, WB7106, and WB7106). 2. Blotted membrane. 3. Primary antibody. 4. Sterile distilled water. 5. Clean dishes.
194
Lars A. Petrasovits
6. Forceps for handling membranes. 7. Orbital shaker, set to 1 rev/min. 8. X-ray film, dark room, and film development chemicals. 9. Autoradiography cassette (Kodak X-OMAT AR or equivalent).
3
Method
3.1 Sample Collection and Preparation Using Trizol®
Caution: When working with TRIZOL Reagent use gloves and eye protection (shield, safety goggles). Avoid contact with skin or clothing. Use in a chemical fume hood. Avoid breathing vapor. TRIZOL® (Invitrogen Cat. No. 15596-026) is a registered trademark of Molecular Research Center, Inc. A recipe for Trizol is provided (Table 2). Unless otherwise stated, the procedure is carried out at 15–30 °C, and reagents are at 15–30 °C. 1. Grind Tissue under Liquid Nitrogen with a mortar and pestle until a fine powder is obtained. 2. Using a cold spatula, weigh out up to 100 mg of frozen tissue powder into 10 volumes of Trizol (see Notes 1–3). 3. Incubate for at least 5 min at room temperature (RT), then centrifuge for 5 min at 12,000 × g. Transfer supernatant to a fresh tube. 4. Add 0.2 volumes of chloroform and shake vigorously for 15 s and incubate for 2–3 min (see Note 4). 5. Remove the aqueous phase (ca. 60 % of total volume) containing RNA. The RNA is precipitated with 0.5 volumes
Table 2 Homemade recipe for 1 L of Trizol
Reagents
Volume/mass
Final concentration
Acid Phenol
380 mL
38 %
Guanidine thiocyanate
94.53 g
0.8 M
Ammonium thiocyanate
30.45 g
0.4 M
Sodium acetate, pH 5.0
33.4 mL of 3 M stock
0.1 M
Glycerol
50 mL
5%
RNase-free water
Adjust final volume to 1 L
Source: http://ipmb.sinica.edu.tw/microarray/newprotocol/TRI.pdf
Protein Blotting
195
Isopropanol per volume Trizol, centrifugation at 12,000 × g, 4 °C and stored under 75 % ethanol at ≤20 °C. 6. Add 0.3 volumes ethanol per volume Trizol used for homogenization to the organic phase. Mix samples by inversion, incubate at RT for 3 min and centrifuge at
