Week 2: SDS-polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blotting

Introduction

Polyacrylamide gel is a 3D network of polymerizing acrylamide and the crosslinker, Bis (N, N'-methylene-bisacrylamide) (Figure 5-3).¹⁷ Polymerization is initiated from ammonium persulfate (AP) as a free-radical source and tetramethylethylenediamine (TEMED) as a catalyst, which activates acrylamide monomer (Figure 5-3a). Although less common, riboflavin (or riboflavin–5'–phosphate) is also a source of free radicals. Combining with bisacrylamide as crosslinkers, the linear polyacrylamide chains form a 3D web (Figure 5-3b). There are factors affecting the rate of acrylamide polymerization. In addition to concentrations of monomer and catalyst, increasing temperature (optimal temperature 23–25°C) drives the polymerization reaction faster and results in early chain termination, leading to non-uniform pore structures. A low temperature makes the gel porous and turbid. Both non-optimal temperatures produce rigid gels. The presence of oxygen traps free radicals and oxidizes TEMED, therefore inhibiting polymerization. Low pH protonates TEMED, decreasing its catalytic activity and thus slowing polymerization.

The pore size of polyacrylamide gels is determined by total monomer concentration (%T, in g/100 ml) and weight percentage of crosslinker (%C).

[latex]\text{\% Total solids content (\%T) } = \frac{g(\text{acrylamide} + \text{bisacrylamide}) \times 100\%}{100 \,\text{ml}}[/latex]

[latex]\text{\% Crosslinker to total acrylamide (\%C) } = \frac{g(\text{bisacrylamide}) \times 100\%}{g(\text{acrylamide} + \text{bisacrylamide})}[/latex]

Both %T and %C can be optimized to best separate and resolve different sized molecules. The %T indicates the relative pore size of the resulting polyacrylamide gel. A higher %T refers to a larger polymer-to-water ratio and smaller pores on average. The acrylamide monomer stock solutions are made as 30–40%. Acrylamide monomer-to-bis ratios determine the type of molecules to resolve in the gel. The acrylamide: bisacrylamide ratios 19:1, 29:1, and 37.5:1 solutions represent a crosslinking percentage of 5% for separating denatured DNA and RNA, 3.3% for native DNA and RNA, and 2.6% for protein, respectively.¹⁷

The combination of gel pore size and protein charge, size, and shape determines the migration rate of the protein. The denaturing SDS polyacrylamide gel electrophoresis (SDS-PAGE) separates proteins based on their sizes.^17,18 SDS, the anionic detergent, binds to the proteins (one SDS molecule for every two amino acids). This causes the polypeptides to denature by eliminating the secondary and non-disulfide-linked tertiary structures, imparting a large negative charge on the proteins to mask any charge present, and distributing negative charges surrounding the protein molecules. Thus, proteins migrate toward the positively charged anode. In addition to SDS, in so-called reducing SDS-PAGE, proteins may be briefly heated to near boiling in the presence of a reducing agent, i.e., dithiothreitol (DTT) or β-mercaptoethanol (β-ME), to further denature the proteins by reducing disulfide linkages and thus removing the tertiary (folding) and quaternary (multimeric) structures. Reducing SDS-PAGE is used to separate proteins based on their sizes.³ There is an inversely linear relationship between log molecular weight and its migration distance or retention factor (Rf) (Figure 5-4). The proteins are completely denatured and thus cannot be used to analyze native proteins, whose biological activity must be retained for subsequent functional tests. Non-denaturing/non-reducing PAGE, also called native gel electrophoresis, may be used when protein native structure is required for further analysis (e.g., enzyme activity). For native gel electrophoresis, the proteins are not denatured with SDS or reducing agents, and separated based on their charge-to-mass ratio.

Two types of polyacrylamide gel are discontinuous and gradient gels.¹⁸ SDS-polyacrylamide discontinuous gel electrophoresis is the most commonly used system. It concentrates the protein sample into a small volume with a stacking gel on the top, increasing the gel's resolution. The gel is made and run in a glycine-containing buffer. The top stacking gel (4% acrylamide, pH 6.8), which is at a pH where most glycine molecules attain a neutral charge (Figure 5-5).¹⁹ The negative chloride ions move faster, followed by the negatively charged proteins and then the neutral glycine ions at the tail end. Thus, the proteins stack between the chloride and glycine. Due to the large pore size and the voltage gradient, the proteins quickly move through the stacking gel and form a tight band as they reach the resolving (separating) gel, where pore size decreases significantly. After entering the resolving gel, where the pH increases, glycine becomes negatively charged and travels faster behind the chloride ions, leaving proteins being separated at the tail end of the resolving gel.

The other type of gel is a gradient gel (pH 8.2), where proteins are compressed (stacked), move through a pore gradient, and gradually separate due to a frictional resistance increase of the gel matrix. Unlike discontinuous gel, stacking and unstacking occur continuously in the gradient gel and thus deposit each protein at a different position. For protein separation, commercially available precast gels are optimized for different effective separation ranges of denatured and native proteins (Table 5-3).

After SDS-PAGE, proteins in the gel can be visualized with a staining reagent. Ponceau S detects protein levels at 200 ng and higher, which can be used with gels and membranes, such as polyvinylidene difluoride (PVDF), nylon, and nitrocellulose (NC).²⁰ It is a reversible staining dye, and the stained membrane can be used for further immunodetection. In contrast, Coomassie Brilliant Blue/GelCode Blue (Safe) Protein Stain, a Coomassie G-250 dye-based reagent, is only compatible with gel and PVDF membranes but can detect protein levels at 50 ng and higher.²¹ Depending on the type of sample and testing, a higher sensitivity detection stain, i.e., Coomassie blue, is preferred. However, these stains can inhibit subsequent immunodetection, as the alcohols and acids in the solution cause the fixation of the protein samples within the gel/membrane after use and are, therefore, inappropriate for immunodetection following PAGE.²²

Western blot analysis identifies and locates specific proteins in a protein mixture based on their ability to bind to particular antibodies after immunodetection.^22,23 It can provide information on protein size by comparing it to a size marker or ladder and protein expression by comparing it to the control, i.e., an untreated sample or another cell type or tissue. The target protein's size and the membrane selection are critical factors affecting the detection sensitivity of proteins in a Western blot analysis. One of the major steps in the Western blot analysis is to transfer proteins from the polyacrylamide gel to the NC, PVDF, or nylon membrane after electrophoresis so that specific proteins can be detected by immuno-detection techniques. The selection of an electroblotted membrane is one of the key factors affecting the detection sensitivity of the protein transferred from the gel to membrane during Western blotting. The binding capacity of the NC membrane to low molecular weight proteins (~ 20 kD) is better than that of the PVDF membrane.²⁴ In contrast, the PVDF membrane has a better capacity to bind to high molecular weight proteins (~150 kD) and glycoproteins than the NC membrane. There is no significant difference in binding capacity between the two membranes with protein sizes between 50 and 80 kD. However, Kurien et al. (2015) compared the binding ability of bovine serum albumin (BSA, Mw 66.5 kD) to these two membranes, finding that the amount of BSA transferred to the PVDF membrane was greater than that of the NC membrane (170 µg vs. 80 µg BSA bound/cm²).²³ In addition to having a greater mechanical strength over NC membrane, the advantages of using PVDF membranes include (a) being compatible with commonly used stains with low backgrounds, high sensitivity, and high binding capacity, (b) eliminating possible protein "blow through" and improving retention on the membrane under harsh conditions, i.e. in the presence of organic solvents or under acidic or basic conditions, and (c) being resistant to solvents and therefore able to be stripped and reprobed.²⁴

There are three major electrophoretic transfer systems for wet, semi-dry, and dry blotting methods.²⁵ The differences among the three transfer methods are mainly the transfer buffer requirements and transfer time, from 30–120 mins, 10 mins, and 3 mins for wet, semi-dry and dry, respectively. As technology advances, automated Western blot processors are becoming commercially available. These benchtop instruments automatically perform the hands-on steps of Western blot processing, including blocking, washing, and antibody incubations.²⁶

For this lab exercise, protein samples prepared from last lab exercise, including proteins from fruit fly hemolymph, BCS, and FBS, are separated by reducing SDS-PAGE and then transferred onto a PVDF membrane. The gel is then stained with GelCode, and the membrane is with Ponceau S (optional). The membrane obtained will be used for immunodetection in the next lab exercise.

Procedure

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

A. SDS-Polyacrylamide discontinuous gel preparation

Note: Although we use the pre-cast SDS-polyacrylamide mini-gels, you need to know how to make the discontinuous gel following the protocol below.

1. Assemble gel plates with 1.5 mm spacers.
2. Prepare and pour a separating gel.
   1. Add the following to a 25 ml sidearm flask:
      

      Units	Ingredients
      3 ml	30%:0.8%::acrylamide:bisacrylamide
      2.25 ml	1.5 M Tris-HCl, pH8.8
      0.09 ml	10% SDS
      3.6 ml	H2O

   2. Degas the gel mixture for 10–30 sec to remove O₂, which interferes with acrylamide polymerization by reacting with the free radicals SO₄^-, and add:
      

      Units	Ingredients
      45 µl	10% ammonium persulfate (AP)
      4.5 µl	100% tetramethylethylenediamine (TEMED)

   3. Swirl to mix gently and add to the gel sandwich using a Pasteur pipette.
   4. Prepare a solution of 1 µl TEMED and 10 µl 10% AP in 2 ml H₂O, immediately overlay the solution on top of the separating gel, and allow the gel to polymerize for 1 hr.
3. Remove the overlay from the separating gel using a Whatman filter paper.
4. Prepare and pour a stacking gel.
   1. Add the following to a 5 ml sidearm flask:
      

      Units	Ingredients
      0.4 ml	30%:0.8%::acrylamide:bisacrylamide
      0.75 ml	0.5 M Tris pH 6.8
      0.03 ml	10% SDS
      1.82 ml	H2O

   2. Degas for 5–10 sec and add:
      

      Units	Ingredients
      15 µl	10% AP
      1.5 µl	100% TEMED

   3. Swirl to mix gently and pour into the gel sandwich using a Pasteur pipette
5. Insert the comb and allow polymerizing for 45 minutes.

B. Protein separation by SDS Polyacrylamide Gel Electrophoresis (PAGE)

1. For every two groups (1 & 2, 3 & 4, or 5 & 6), place two precast gels in one electrophoresis apparatus following the manufacturer's instructions. After electrophoresis, one gel will be used for Western transfer (see section C) and the other for staining with GelCode (see section D).
2. Fill the apparatus with running buffer (0.025 M Tris-base, 0.192M glycine, 0.1% SDS), using 125 ml in the top chamber and 200 ml in the bottom chamber.
3. Flush each well with a running buffer using a 1 ml micropipette.
4. Load 5 µl of protein molecular weight markers (M), followed by 15 µl of each protein sample (2.5–0.025 µg) and Drosophila hemolymph (7.5 µg) into each corresponding well (Table 5-4).
5. Electrophorese the samples at 200 V for 35 mins until the bromophenol blue tracking dye is at the bottom of the separating gel.

While waiting for electrophoresis to complete, prepare the PVDF membrane by prewetting it in methanol and then transferring it into the blotting buffer for 15–20 mins.

C. Western transfer

After electrophoresis, remove the gel plates from the apparatus.

1. For transblot, disassemble one of the gels and transfer it into a plastic container with 30–50 ml of blotting buffer (0.025M Tris, 0.192 M glycine, 20% methanol, pH 8.3) to equilibrate for 20 mins on a shaker. During gel disassembly, it is better to keep the gel on the large plate and then carefully dislodge the gel side down from the glass plate into the buffer so that the first well will be on the left side of the gel. Avoid tearing the gel by wetting your fingers before touching it.
2. Assemble the gel into the blotting holder for Western blotting by following the manufacturer’s instructions. Here, we follow the instruction for the Bio-Rad Criterion Blotter for Western transfer (Figure 5-6). Ensure there are no air bubbles among the gel, membrane, and blotting papers. Pay special attention to the gel orientation in the assembly.
   

   

3. Slide the assembly holder into the slot in the blotting chamber. The chamber should contain about 1/2 liter of the blotting buffer. Ensure that each holder's membrane is in the same orientation.
4. Place the frozen Bio-Ice in position and continue filling up the blotting buffer.
5. Connect the lid and electrodes to the apparatus with the positive electrode on the same side as the membrane.
6. Blot at 100 V for 45 min or until the ice completely melts.
7. Turn off the power and carefully disassemble the gel from the cassette. Mark wells on the membrane with a pencil before removing the gel. Ensure no gel piece is stuck on the membrane.

D. Gel staining with GelCode

1. Transfer the other gel to a large 50 mm Petri dish with 30–50 ml reverse osmosis (RO) water and place the dish on a shaker at room temperature for 5 min.
2. Replace the RO water with a fresh 50 ml RO water and place the dish on a shaker for another 5 min.
3. Repeat step 2.
4. Replace the RO water with 20–30 ml GelCode and put it back on a shaker for 1 hour.
5. Drain the staining reagent into a designated waste container.
6. To destain, cover the gel with 50 ml of RO water, and shake for 30 min.
7. Repeat step 2 again.
8. Discard the RO water, fill the dish with 50 ml RO water, and place the dish in the refrigerator overnight for a sharper image.
9. The next day, take the gel out of the dish and place it on plastic wrap on top of white paper before taking a picture.

E. Membrane staining with Ponceau S (Optional)

These steps show that Ponceau S reversibly stains proteins on the membrane after electrophoresis and transblotting (from Procedure C, above).

1. After marking the wells and removing the gel, place the membrane containing transferred proteins in a 50 mm Petri dish and rinse the membrane with 50 ml RO water.
2. After discarding the water, soak it with Ponceau S stain (0.2% Ponceau S, 3% trichloroacetic acid (TCA), 3% sulfosalicylic acid). Allow it to stand for 10 minutes.
3. Pour the excess stain back into its container. Rinse the membrane with distilled/RO water until no more stain washes off. The proteins should be pink.
4. To record the pattern of Ponceau S staining, take a picture of the membrane. Put a ruler next to the marker (lane 1) while photographing.
5. Place the membrane in a plastic container with wet Kimwipes to keep it moist and store it at 4°C.

Data Analysis and Discussion

1. What is the % of acrylamide, excluding bisacrylamide, in the (a) stacking and (b) separating gel described in the protocol? (Ignore the volume of AP and TEMED.)
2. A standard curve constructed based on the electrophoresed protein molecular weight marker in the gel can determine the sizes of major proteins after electrophoresis.
   1. Make a standard curve by graphing the log₁₀ Mw of each protein (Y-axis) against the migrating distance or Rf value of each protein of BioRad Precision™ Plus Protein Dual Color Standards (see Figure 5-7) in the M lane (X-axis).
   2. Use the equation derived from the standard curve to estimate the Mw of major bands in the stained (i) gel and (ii) membrane.