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SDS-PAGE and Western Blotting Lab report (extensive methods section)

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Introduction

BM310 Generic Skills Labs | Lab Report 1 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis and Western Blotting. Abstract This experiment made use of the Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis technique to plot a curve displaying the electrophoretic mobilities of 7 proteins against the known molecular weights. Another sample was also run in the SDS-PAGE but with an unknown protein sample. Two proteins were found in the sample and their electrophoretic mobilities alongside the standard curve made with the known proteins, were used to determine the molecular weight of these proteins. Western Blotting was carried out with the separating gel from the SDS-PAGE and the proteins were observed on a nitrocellulose membrane, achieved by several procedures, including treatment with antibody solution and a colour development solution, to ensure the protein could be visualised. _____________________________________________________________________ Introduction Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is a common biomolecular technique used to separate protein mixtures by exploiting their different electrophoretic mobilities. Electrophoretic mobilities differ in proteins according to a number of factors including chain length, molecular weight, the way the protein folds into its tertiary and/or quaternary structures, and the number and type of bonds that are involved in this high order protein folding. Because this technique is used for separating proteins, it is one of the most common procedures carried out in many fields, including biochemistry, forensics, molecular biology and genetics. The technique mixes the protein with sodium dodecyl sulphate (SDS) ...read more.

Middle

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. ...read more.

Conclusion

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). ...read more.

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