This experiment set out to separate a sample of known proteins and a standard curve be plotted in order to estimate the molecular weight of unknown protein samples. This unknown protein was then subject to the western blot technique using colorimeric detection with the aid of phosphatase substrates which bind to an enzyme linked antibody that had been used to tag the protein.
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Methods
SDS Gel Electrophoresis
This experiment used the Laemmli discontinuous buffer system, which is the most commonly used procedure for SDS gel electrophoresis. This technique uses a discontinuous gel consisting of a resolving gel and a separating gel, both of which had to be made prior to the start of the experiment. A 7.5% separeting monomer gel solution was made by combining 4.85mL of distilled water, 2.5mL of 1.5M Tris-HCl (pH 8.8), 100µL of 10% (w/v) SDS stock, 2.5mL of a 30% acrylamide mix, 50µL of 10% ammonium persulphate (APS) and 5µL of tetramethylethylenediamine (TEMED). The APS and TEMED were not added until the gel was ready to be poured into the gel sandwich. A comb was placed into the gel sandwich so the positions of the bottoms of the wells could be noted, then it was removed. The gel was completed by adding the APS and TEMED, and the solution mixed well, without shaking to avoid air bubbles being created which would destroy the gel. The gel solution was carefully but quickly (as it would polymerise fairly soon) transferred to the gel sandwich using a Pasteur pipette and bulb, making sure the top of the gel was around a centimetre from where the wells would end in the loading gel. Butanol was poured on top of the separating gel and it was allowed to polymerise.
A 4% stacking gel was then made using 6.1mL of distilled water, 2.5mL of 0.5M Tris-HCl (pH 6.8), 100µL of 10% SDS stock, 1.3mL of 30% acrylamide solution, 50µL of 10% APS and 10µL of TEMED. The butanol was then removed from the top of the pre-prepared resolving/separating gel and the gel sandwich was washed with water to remove the excess butanol. The stacking gel was then poured carefully on top of the resolving gel and the comb inserted. This gel was then allowed to polymerise for 45 minutes and then the comb was removed, and washed with distilled water once more. Pre-stained protein standards and a pre-stained unknown protein sample were used in this experiment. Both sample were transferred into microcentrifuge tubes and incubated at 100°C for 3 minutes to denature the proteins and for the anons of SDS to bind to the protein (giving it a negative charge proportional to the molecular weight of the protein). These protein samples were then loaded into wells made in the stacking gel by the comb, and the electrophoresis bath was filled with buffer and closed. 200 volts was applied to the gels for around 45 minutes, by which time, the blue band was 1cm from the bottom of the resolving gel. The distance of each of the coloured bands was recorded. The glass plate covering the gel was removed so the gel could be removed for the second stage of the procedure, western blotting.
Western Blotting
When the gel was remobed from the glass, it was soaked in transfer buffer, whilst 2 fibre pads were also saturated in buffer. A piece of nitrocellulose paper was cut to the same dimentions of the gel, as were pieces of thick blotting paper. The nitrocellulose was wet with the transfer buffer by capillary action, however it was not flooded quickly as this woulc cause air bubbles to form in the matrix, which would block the transfer of molecules to the membrane from the gel.
The blotting apparatus is a Trans-Blot tank with slots for ‘cassettes’ which is two panels which hold together the sandwich of fibre pads, blotting paper, the gel and the nitrocellulose membrane. One of the fibre pads was placed on the grey panel of the holder and a saturated piece of blotting paper was put on top of this. The gel, which has been equilibrated in buffer is placed on top of the filter paper. The pre-wetted nitrocellulose transfer membrane was then placed on top of the gel by holding at both ends and lowering from the middle, to reduce the risk of air bubbles. Any remaining bubbles were removed by rolling a test tube along the length of the membrane, forcing the bubbles out at the end. A final piece of saturated blotting paper was layered on top, again, avoiding air bubbles. The second fibre pad was put on top along with the other panel of the cassette. The cassette was then placed into the tank with the grey panel at the cathode side of the tank, which is then filled with transfer buffer. The red cable was then plugged into the black power outlet and the black cable in the red outlet. This is because when an acidic buffer is used, the transfer is anode to cathode, but the polarity would have to be reversed as the protein will be going from the cathode side to the anode side. Blotting was carried out at 30v, 0.1A overnight.
Identifying Proteins
The blotted membrane was placed in the staining bath and covered with blocking solution for 10 minutes. 5mL of antibody solution was added and was incubated at room temperature for 30 minutes. The membrane was then washed with 10mL of time tagged buffer solution (TTBS) for 5 minutes, and repeated a further two times. The buffer was poured off and 5mL of Rabbit IgG Alkaline Phosphate solution was added and incubated, shaking gently throughout, for 30 minutes at room temperature. This was washed a further 3 times in TTBS. 10mL of a colour developing solution was added and it was incubated in the dark for around 15 minutes, until a colour had appeared. The reaction was halted by rinsing the membrane with distilled water and it was dried on filter paper.
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Results
The SDS-PAGE separated the proteins according to their size; with the smallest protein (Triosephosphate Isomerase from rabbit muscle) at 26.6kDa travelling 4.5cm and the largest protein (Macroglobulin from human plasma) at 180kDa travelling only 1.5cm through the separating gel. Table 1: Protein Standards, shows the 7 known protein standards. The unknown protein sample revealed two proteins, protein A, which travelled 2.6 cm and protein B, which travelled 4.3cm. This information was used alongside a standard curve (Figure 1: Standard Curve of Known Proteins) to estimate the molecular weight of unknown protein A and unknown protein B. Protein A was estimated to be around 63kDa and protein B just under 28kDa. These results are displayed in Table 2: Unknown Protein Sample and plotted on Figure 1. Also below, is Figure 2: Photograph from Western Blotting which shows the result of the western blotting technique. The protein transferred to the nitrocellulose and is clearly visible after the several treatments using antibody solutions.
Table 1: Protein Standards. This table shows the 7 different proteins present in the known standard solution. They are listed, with their corresponding molecular weights, log10 molecular weights and the distance they migrated down the SDS gel.
Table 2: Unknown Protein Sample. This table shows the unknown proteins, A and B which were present in the unknown sample. Also represented, are their molecular weights, their log10 molecular weights, and the distance they migrated down the SDS gel. They are plotted on Figure 1: Stardard Curve of Known Proteins, alongside the standard curve of known protein samples.
Figure 1: Standard Curve of Known Proteins. This is a representation of the 7 protein standards that were used in an SDS gel electrophoresis experiment. It shows proteins ranging from 26.6kDa to 180kDa, which migrated between 1.5 and 4.5 centimetres down the SDS gel. Also plotted on the graph are Protein A and Protein B, which migrated 2.6 and 4.3cm respectively.
Figure 2: Photograph from Western Blotting. This is a photograph taken of the result of the western blotting test which was done to transfer the protein from the separating gel, to a nitrocellulose membrane, which underwent a series of treatments (see Methods), so that the protein could be visualised on the membrane.
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Conclusion
This experiment demonstrated that sodium dodecyl sulphate polyacrylamide gel electrophoresis is an accurate technique for separating proteins as its principle function is to standardise the mass to negative charge ratio. This ensures that the proteins are being separated incrementally due to their mass and not according to their isoelectric points. It also showed that Western Blotting an effective analytical technique in probing proteins for detection by using antibodies specific for that protein.
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Discussion
The results from this experiment showed that there were 2 unknown proteins in the unknown sample. These were successfully separated using SDS-PAGE, and their molecular weight was estimated using a standard curve that was plotted of distance migrated (cm) vs log10 of the known molecular weights. This curve was then used with the distance migrated data of the unknown sample to estimate the molecular weight of the unknown proteins. Protein A was estimated to have a mass of 63.1kDa and protein B, 27.9kDa.
It was reported that the molecular weights of proteins could be estimated with at least 10% ± the actual value (Weber, 1969), however this experiment used around 40 proteins for its standard curve, reducing the margin of error compared to this study, significantly. Because this study only used 7 proteins for a standard curve (largely due to time and budget constraints), this significantly reduces the reliability of the estimates of Protein A and Protein B. To improve reliability, the same 7 proteins could undergo several SDS-PAGE experiments or the sample size could be increased.
A similar technique to SDS-PAGE is Native-PAGE, whereby the separation occurs without denaturing the protein. Because the proteins are not denatured, they are less predictable in the way they move through the gel, and take much longer. Different proteins also have different charges and different strengths of charges, so some proteins which may be larger although have a strong negative charge, may travel further than a smaller protein with a slightly positive charge. This can make Native-PAGE unhelpful when trying to differentiate proteins by their size. SDS technique overcomes this problem by imparting a negative charge on the protein relative to its size, giving almost all proteins uniform ratio of mass to charge. Proteins will then be separated incrementally according to their size and not its individual isoelectric point (Shapiro et al, 1967).
The Western Blot method has been a popular technique since its discovery in the 1980s (Burette, 1981). Other than nitrocellulose other membranes may be used for protein transfer from polyacrylamide gels. These include polyvinylidene fluoride and DBM-paper. These may be used in place of nitrocellulose for the classes of protein that do not bind to nitrocellulose (Towbin et al, 1979). Comparisons between the Towbin study and the study by Renart et al, show that nitrocellulose has a much greater efficiency of proteins transferred to the membrane compared to DBM-paper (Towbin et al, 1979 & Renart et al, 1979).
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References
W. Neal Burnette (April 1981). "". Analytical Biochemistry (United States: Academic Press) 112 (2): 195–203.
Renart J, Reiser J, Stark GR (1979). "Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.". Proc Natl Acad Sci U S A. 76 (7): 3116-3120.
Shapiro AL, Viñuela E, Maizel JV Jr. (September 1967). "Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.". Biochem Biophys Res Commun. 28 (5): 815-820.
Towbin H, Staehelin T, Gordon J. (1979). "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.". Proc Natl Acad Sci U S A. 76 (9): 4350-4354.
Weber K, Osborn M (August 1969). "The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis.". J Biol Chem. 244 (16): 4406-4412.
