Created: Sep 2021
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
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
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
wash laboratory equipment with detergent
prepare 10% (w / v) sodium dodecyl sulfate (SDS) solution (6 ml distilled water + 0.6 g SDS)
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)
prepare transfer buffer (dissolve Tris 3.0 g and 14.4 g glycine in 1,000 ml of distilled water)
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)
wash the silicon rubber packaging, sample comb and the glass plates
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
press the silicone rubber packing along the edges of one gel plate, attach the second plate to it and fix with a clip
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
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
add 31.25 ul of 10% ammonium persulfate and 3.15 ul of TEMED and mix by inverting the tube
pour this solution into the gel rack up until the horizontal line
pour distilled water on top of the gel, filling up the rack, wait until the gel solidifies
cover the top of the glass plates with plastic wrap (to avoid water evaporation), store in refrigerator
Preparation of the protein sample
place ice in a 500 ml beaker
place protein samples (homogenate, membrane lysate, soluble fraction) and SDS-PAGE sample buffer on the ice
mix the samples with SDS-PAGE sample buffer, making three 50 μl protein sample solutions, each containing 100 μg of protein
heat at 100°C for 5 min
store in a freezer
Preparation of the stacking gel
retrieve the gel rack with 10% separating gel from the refrigerator
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
remove the water (top layer) from the gel rack
add 15.7 μl of 10% ammonium persulfate and 3.15 μl of TEMED to the gel solution, mix
pour the gel solution into the gel rack, on top of the separating gel
put a comb in the rack and let the gel solidify
SDS-PAGE of Protein Samples
removing the sample comb and the clips from the gel rack, take off the silicone rubber packing
fill the migration tank with the electrophoresis buffer
clip the gel plates to the electrophoresis apparatus
remove the air bubbles with a syringe, wash the insides of the wells
add 5 μl of sample buffer to lanes 1,6 and 10 and 5 μl of molecular weight marker to lane 2
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
connect the electrode wire to the power supply, pass 0.04 A current
cut the PVDF membrane into a 10 x 7.5 cm sheet
place 40 ml of methanol in tapper ware and shake the membrane in the solution
after 10 min, remove the methanol, pour in the transfer buffer and continue shaking
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
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)
place the gel holder cassette (black side down) on the vat
open the cassette and place the fiber pad dampened with transfer buffer on the black side
pour the transfer buffer into the tapper ware, put the gel in the buffer (cut on the right)
place the gel on filter paper dampened with transfer buffer, put them on the fiber pad
place the PVDF membrane on top of the gel (no air bubbles)
put filter paper moistened with transfer buffer on top, fiber pad on top of that, and close the casette
place the transfer device in an ice box placed on top of magnetic stirrer, put one magnetic bar inside
insert the gel holder cassette into the transfer device holder, black sides aligning
after the cooling unit is placed in the transfer apparatus, it is filled with transfer buffer
put on a cover and connect the electrode wire to the power supply and apply 100 V for 45 minutes
cut the power supply, remove the wires
open the gel holder cassette and remove the fiber pad and filter paper
make 2 inclined cuts in the at the bottom of lane 1 and 6 of the transfer membrane,
cut the membrane with scissors across lane 1, 6 and 10, making 2 fragments
shake the transfer membrane fragment containing the molecular weight marker (corresponding to lanes 2-5) in the CBB solution for 5 minutes
place the transfer membrane in destaining solution
decolorize the background’s blue colour until the protein bands are clearly visible
place the other transfer membrane fragment in transfer buffer, store in refrigerator
2.3. Binding Antibodies
block the transfer membrane fragment (corresponding to lanes 7-9) is with 1% BSA solution for 1 h at room temperature
wash (shake) the membrane with 30 ml of TBS-Tween 20 for 5 min, 3 times
put the membrane between 2 vinyl sheets and seal it with a polysealer, leaving one shorter side open
add 1 ml of primary antibody solution (mouse anti-GAPDH antibody) and spread it across the membrane
remove bubbles, close the vinyl bag with the polysealer
tape the bag to a flat support plate and leave for 1 h
wash the membrane with 30 ml of TBS-Tween 20 for 5 min, 3 times
put the membrane between 2 vinyl sheets and seal it with a polysealer, leaving one shorter side open
add 1 ml of secondary antibody solution (alkaline phosphatase (AP) labelled anti-mouse IgG antibody) and spread it across the membrane
remove bubbles, close the vinyl bag with the polysealer
tape the bag to a flat support plate and leave for 30 min
wash the membrane with 30 ml of TBS-Tween 20 for 3 min, 5 times
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
put the membrane (protein transfer side up) on a plastic wrap
place all of the AP chromogenic substrate solution on the membrane, wait until the colour develops
put the membrane in water to stop the reaction
replace the water, wash the membrane, then allow it to dry
determine the molecular weight of the protein detected by the antibody
Three protein samples were used in SDS-PAGE. However, the homogenate sample has dried out, probably due to improper closing of the microtube in which the liquid was stored. Therefore, a homogenate sample borrowed from Group 7 has been used in this experiment.
After performing SDS-PAGE, the proteins were transferred from the gel to PVDF transfer membrane. The lanes 2-5, which contained the molecular weight marker’s lane, were stained with CBB, which resulted in blue bands appearing in the marker’s and protein samples’ lanes (Figure 1).
As we can see from Figure 1, all 3 protein samples’ migration patterns have plethora of bands, which shows that there are many types of proteins is each of the 3 samples. For all 3 samples, most of the protein bands appear between the Marker’s 1st and 6th band, which corresponds to 31- 200 kDa molecular weight. This shows that most of the proteins in the samples are within this weight limit.
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.
In the next part of the experiment, we tried to detect GAPDH protein by binding antibodies and treating the membrane (lanes 7-9) with AP chromogenic substrate. As the result, one band appeared in each of the three lanes (Figure 3). The migrations of all three bands are the same (26.5 mm), which means that they correspond to the same molecular weight. Thus, it is also likely that they correspond to the same protein- GAPDH. Since we determined the migration of the protein band, we can use the best fit line (curve) in Figure 2 to find out the molecular weight of the protein from Figure 3 (find what molecular weight corresponds to 26.5mm migration).
Molecular weight of GAPDH = 35.4 kDa
As GAPDH was detected in all three (homogenate, membrane lysate and soluble fractions) protein samples, we can say that GAPDH is probably omnipresent in rat’s liver. Since it was detected in membrane lysate, it can be deduced that GAPDH is one of the constituents of cell membrane. Moreover, its presence in soluble fractions sample suggests that it is water soluble.
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.
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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