Western Blotting

Created: Sep 2021

1. Objective

Proteins are major components of the organism and play an important role in various biological phenomena. It is estimated that about 100,000 kinds of proteins exist in our body. Thus, is problematic to find the protein of interest and study its structure and function among such a large number of protein clusters. SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which is a method of separating and aligning proteins by molecular weight, is the first step in analysing protein samples

In this experiment, SDS-PAGE is performed using rat liver-derived homogenate, membrane lysate, and soluble fractions as protein samples. Subsequently, western blotting is performed to transfer these proteins to a polyvinylidene difluoride (PVDF) membrane. The transfer membrane is reacted with a primary antibody that binds to a specific protein (GAPDH), and then reacted with an enzyme-labeled secondary antibody that binds to the primary antibody. The colour is developed using the enzyme substrate, and the protein can be detected.

The purpose of this experiment is to learn the techniques of SDS-PAGE and Western blotting and understand how to detect proteins using antibodies.

2. Materials and Methods

2.1. Preparation

Prepare 30% acrylamide-bis solution, 1.5 M Tris-HCl buffer (pH 8.8), 0.5 M Tris-HCl buffer (pH 6.8), 10% ammonium persulfate (APS), tetramethylethylenediamine (TEMED), SDS-PAGE sample buffer, primary and enzyme-labeled secondary antibody solutions.

Composition of the solutions:

• SDS-PAGE sample buffer: 4.0 ml distilled water, 1.0 ml of 0.5 M Tris-HCl buffer (pH 6.8), 0.8 ml glycerol, 1.6 ml of 10% SDS, 0.4 ml 2-mercaptoethanol, 0.2 ml of 0.5% BPB

• Coomassie Brilliant Blue (CBB) solution: 0.1 g CBB-R250, 65 ml distilled water, 10 ml of acetic acid, 25 ml methanol + filtration

• Decolorizing solution:

10% acetic acid- 40% methanol: 250 ml distilled water, 50 ml acetic acid, 200 ml methanol

10% acetic acid- 25% methanol: 325 ml distilled water, 50 ml acetic acid, 125 ml methanol

Washing laboratory equipment and reagent preparation

1. wash laboratory equipment with detergent

2. prepare 10% (w / v) sodium dodecyl sulfate (SDS) solution (6 ml distilled water + 0.6 g SDS)

3. prepare electrophoresis buffer (dissolve 1.5 g Tris and 7.2 g glycine in 500 ml of distilled water, add 5 ml of 10% SDS solution to the 495 ml of the Tris-glycine solution)

4. prepare transfer buffer (dissolve Tris 3.0 g and 14.4 g glycine in 1,000 ml of distilled water)

5. prepare 1% (w / v) bovine serum albumin (BSA) solution (dissolve 0.4 ml of BSA in 40 ml of TBS)

Gel preparation rack (wear gloves)

1. wash the silicon rubber packaging, sample comb and the glass plates

2. wipe the gel plate (one set), silicone rubber packing, and sample comb with 70% ethanol dampened degreaser, and wipe off excess moisture with a Kimwipe

3. press the silicone rubber packing along the edges of one gel plate, attach the second plate to it and fix with a clip

4. place a sample comb on the gel preparation rack and draw a 1 cm horizontal line 7 mm below the lower end of the comb

Preparation of 10% separation gel

1. mix 2.49 ml of distilled water, 2.10 ml of 30% acrylamide bis, 1.58 ml of 1.5 M Tris-HCl buffer (pH 8.8), 62.5 μl of 10% SDS in a 15 ml tube with a lid

2. add 31.25 ul of 10% ammonium persulfate and 3.15 ul of TEMED and mix by inverting the tube

3. pour this solution into the gel rack up until the horizontal line

4. pour distilled water on top of the gel, filling up the rack, wait until the gel solidifies

5. cover the top of the glass plates with plastic wrap (to avoid water evaporation), store in refrigerator

Preparation of the protein sample

1. place ice in a 500 ml beaker

2. place protein samples (homogenate, membrane lysate, soluble fraction) and SDS-PAGE sample buffer on the ice

3. mix the samples with SDS-PAGE sample buffer, making three 50 μl protein sample solutions, each containing 100 μg of protein

4. heat at 100°C for 5 min

5. store in a freezer

2.2. SDS-PAGE

Preparation of the stacking gel

1. retrieve the gel rack with 10% separating gel from the refrigerator

2. mix 1.52 ml distilled water, 0.33 ml 30% polyacrylamide-bis, 0.63 ml 0.5 M Tris-HCl buffer solution (pH 6. 8) and 25 μl of 10% SDS in a 15 ml tube with a lid

3. remove the water (top layer) from the gel rack

4. add 15.7 μl of 10% ammonium persulfate and 3.15 μl of TEMED to the gel solution, mix

5. pour the gel solution into the gel rack, on top of the separating gel

6. put a comb in the rack and let the gel solidify

SDS-PAGE of Protein Samples

1. removing the sample comb and the clips from the gel rack, take off the silicone rubber packing

2. fill the migration tank with the electrophoresis buffer

3. clip the gel plates to the electrophoresis apparatus

4. remove the air bubbles with a syringe, wash the insides of the wells

5. add 5 μl of sample buffer to lanes 1,6 and 10 and 5 μl of molecular weight marker to lane 2

6. add 10 μl (equivalent to 20 ug of protein) of homogenate to lanes 3 and 7, 10 μl of membrane lysate to lanes 4 and 8, 10 μl of soluble fractions to lanes 5 and 9

7. connect the electrode wire to the power supply, pass 0.04 A current

8. cut the PVDF membrane into a 10 x 7.5 cm sheet

9. place 40 ml of methanol in tapper ware and shake the membrane in the solution

10. after 10 min, remove the methanol, pour in the transfer buffer and continue shaking

11. stop the passage of current when the dye in the sample buffer, bromophenol blue (BPB), is 1-2 cm from the bottom edge of the gel

12. remove the gel from the gel plate and make a cut under the molecular weight marker’s lane

Preparation of the transfer membrane and CBB staining (wear gloves)

1. place the gel holder cassette (black side down) on the vat

2. open the cassette and place the fiber pad dampened with transfer buffer on the black side

3. pour the transfer buffer into the tapper ware, put the gel in the buffer (cut on the right)

4. place the gel on filter paper dampened with transfer buffer, put them on the fiber pad

5. place the PVDF membrane on top of the gel (no air bubbles)

6. put filter paper moistened with transfer buffer on top, fiber pad on top of that, and close the casette

7. place the transfer device in an ice box placed on top of magnetic stirrer, put one magnetic bar inside

8. insert the gel holder cassette into the transfer device holder, black sides aligning

9. after the cooling unit is placed in the transfer apparatus, it is filled with transfer buffer

10. put on a cover and connect the electrode wire to the power supply and apply 100 V for 45 minutes

11. cut the power supply, remove the wires

12. open the gel holder cassette and remove the fiber pad and filter paper

13. make 2 inclined cuts in the at the bottom of lane 1 and 6 of the transfer membrane,

14. cut the membrane with scissors across lane 1, 6 and 10, making 2 fragments

15. shake the transfer membrane fragment containing the molecular weight marker (corresponding to lanes 2-5) in the CBB solution for 5 minutes

16. place the transfer membrane in destaining solution

17. decolorize the background’s blue colour until the protein bands are clearly visible

18. place the other transfer membrane fragment in transfer buffer, store in refrigerator

2.3. Binding Antibodies

1. block the transfer membrane fragment (corresponding to lanes 7-9) is with 1% BSA solution for 1 h at room temperature

2. wash (shake) the membrane with 30 ml of TBS-Tween 20 for 5 min, 3 times

3. put the membrane between 2 vinyl sheets and seal it with a polysealer, leaving one shorter side open

4. add 1 ml of primary antibody solution (mouse anti-GAPDH antibody) and spread it across the membrane

5. remove bubbles, close the vinyl bag with the polysealer

6. tape the bag to a flat support plate and leave for 1 h

7. wash the membrane with 30 ml of TBS-Tween 20 for 5 min, 3 times

8. put the membrane between 2 vinyl sheets and seal it with a polysealer, leaving one shorter side open

9. add 1 ml of secondary antibody solution (alkaline phosphatase (AP) labelled anti-mouse IgG antibody) and spread it across the membrane

10. remove bubbles, close the vinyl bag with the polysealer

11. tape the bag to a flat support plate and leave for 30 min

12. wash the membrane with 30 ml of TBS-Tween 20 for 3 min, 5 times

13. mix 1ml 0.1 M Tris-HCl buffer, 100 μl of 5-bromo-4-chloro-3-indoyl phosphate (BCIP) and 100 ul of nitroblue tetrazolium (NBT) to make AP chromogenic substrate solution

14. put the membrane (protein transfer side up) on a plastic wrap

15. place all of the AP chromogenic substrate solution on the membrane, wait until the colour develops

16. put the membrane in water to stop the reaction

17. replace the water, wash the membrane, then allow it to dry

18. determine the molecular weight of the protein detected by the antibody

However, membrane lysate sample (3rd lane) has many clear bands far above the 1st marker’s band (200 kDa), and slightly lighter bands around and after the 6th marker’s band (31 kDa). This tendency of the bands to appear in the upper part of the migration patterns shows that the proteins in the membrane lysate sample are heaver compared to the other 2 samples, some proteins being heavier than 200 kDa.

In case of homogenate and soluble fractions, there are several clear lines below the 7th marker’s line (21 kDa), which suggests that several proteins in these samples are particularly light. Moreover, for soluble fractions sample, the bands are more sparse between the 1st-4th marker’s line, and absent above that region. This hints that many proteins in soluble fraction sample are probably lighter compared to the other 2 samples.

For all 3 protein samples, we obtained a migration pattern with clear, deep blue lines, mostly within the region covered by the molecular weight marker. This shows that the concentration of the protein samples is suitable for their further analysis by binding antibodies, and isolating GAPDH.

There are 9 band in the marker’s pattern instead of expected 7 due to disrupted flow of the protein. However, the last 2 bands can be ignored when plotting the migration vs. molecular weight graph, which will help to find the molecular weight of GAPDH. The migration (l) of the marker was measured starting at the upper edge of the migration region, and the migration was plotted against the known molecular weight corresponding to each of the 7 bands. The graph is shown in Figure 2.

4. Discussion

The theoretical value of the molecular weight of GAPDH is around 36 kDa (subunit). Since the experimental value of 35.4 Da, the relative error is 1.7%. For a first attempt at this multiple-step experimental method, it could be considered to be a low value. Therefore, it can be deduced that the experimental value largely matches the theoretical value, and the experiment has yielded the expected results.

However, contrary to the predictions, the CBB staining yielded 9 bands instead of expected 7 bands for the molecular weight marker. The lowest 2 bands, which were ignored in this experiment when plotting graph in Figure 2, correspond to low molecular weight (below 20.5 kDa). Those 2 bands could perhaps be removed by increasing the electrophoresis time, increasing the bands’ migration, and thus causing them to be “washed off” the separation gel.

The desired amount of protein in each of the 3 protein samples was 100 μg. The volume of the original protein solutions containing that amount of protein was calculated based on the protein concentration determined by BCA method in the previous experiment. Therefore, any random or systematic errors that occurred in the previous experiment also had an effect on this experiment. Discrepancies between the desired weight and actual weight influenced the appearance (shade) of the bands produced by the respective protein samples. Fortunately, this error probably did not influence the position of bands, which means it did not have any effect on the experimental value of GAPDH molecular weight.

5. Theory

The function of SDS and 2-mercaptoethanol

In this experiment, SDS and 2-mercaptoethanol were the ingredients of the SDS-PAGE sample buffer, which was mixed with the 3 protein samples in section 2.1. SDS makes a strong bond with the proteins in the samples (about one SDS molecule per 2 amino acids), denaturing their secondary and tertiary structures. Furthermore, when the protein samples with SDS are boiled, they gain a negative charge that covers the hydrophobic parts of the proteins and thus overcomes the positive charges that are usually present in it. Thanks to this, the charged proteins are able to move through the gel as the current is being applied, their speed dependent on their size (molecular weight).

In addition, 2-mercaptoethanol is added in order to break disulphide bonds by reducing them. It further promotes the denaturation of proteins, breaking up tertiary and quaternary structures.

On GAPDH protein

GAPDH, abbreviation for glyceraldehyde-3-phosphate dehydrogenase, is an enzyme omnipresent throughout the cells of many organisms, including humans. In fact, it is present (in high concentration) in almost all tissues, which is why it is a popular protein for western Blotting procedure.

GAPDH is involved in multiple processes throughout the cell. For example, in the cytoplasm, it participates in the ATP and pyruvate production by anaerobic glycolysis, and other processes associated with energy metabolism. It also takes part in nuclear RNA export, DNA replication and repair, membrane fusion and phosphotransferase activity, as well as iron metabolism, receptor mediated cell signalling and apoptosis. GAPDH is made up of 4 identical subunits. High levels of GAPDH are often associated with cancer, the protein being deregulated in breast cancer, melanoma, pancreatic cancer and many more.

More accurate method of determining the molecular weight of GAPDH

In this experiment, we used denaturing agents that broke down the quaternary structure of GAPDH consisting of 4 subunits, so the molecular weight value that we found corresponds to one subunit. To determine the weight of the entire (native) molecule, instead of using SDS-polyacrylamide gel, we could attempt to use a non-denaturing polyacrylamide gel, which could yield more accurate results. For example, we could perform non-denaturing PAGE using polyacrylamide gel containing 10X TBE buffer, 20% acrylamide, TEMED and 10% (w/v) APS, and use1X TBE to fill up the electrophoresis tank.

Wet blotting and semi-dry blotting

Wet (tank) protein transfer was the method used in this experiment. It provides an efficient transfer in case of both small and large proteins (wide range), especially so in case of the latter. Moreover, it produces sharp, clearly visible bands. However, wet transfer is very time-consuming (1 h - one night) and requires a cooler (ice-box etc.) to prevent buffer breakdown. Moreover, a large quantities of buffer are needed to perform wet transfer.

On the other hand, semi-dry transfer takes way less time (under 1 h) and does not require large buffer volumes. It is a particularly good method for small protein transfers. Unfortunately, it is not a recommended method for large proteins, and has a lower transfer efficiency compared to the first method. It also often results in blurry, indistinct bands.

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