Western Blotting Experiment

Western blotting is a procedure used to detect specific proteins in a given sample. Gel electrophoresis is used to separate the proteins which can be observed as thick and thin bands on the electrophoresis gel. In this experiment we use SDS-free polyacrylamide gel. Sample proteins used in this case are bovine serum, human serum, goat serum, chicken serum and horse serum. Since the SDS is negatively charged, sample proteins move to the positively charged anode through the gel. Small proteins migrate faster since it is light and is seen further below the gel than the larger proteins which migrate slowly and stays at the top of the gel. Therefore, proteins are separated according to their size in gel electrophoresis. A notch is made at the top

A magnetic stirrer was used during western blotting in order to maintain an even temperature as western blotting will cause the temperature to rise which will result in denaturation of the proteins. In this experiment, two gels were used, one which underwent blotting and one without blotting in order to locate the position of human serum albumin which was present in all the samples loaded. In each of the two gels and in the nitrocellulose membrane used, a notch was made to identify the cathode from the anode and to identify the start of the lanes. Also, caution had to be taken while pressing on the nitrocellulose membranes to ensure that the proteins weren’t denatured during the process. Also precautions had to be taken while transferring the contents onto the rod by moving the rod in one direction ensuring maximum transfer of contents to the

This catalyzed the reaction which contributed to the oxidized form of naphthol which in turn formed purple precipitate which helped in determining the location of the human serum albumin. Therefore, absence of purple bands on the nitrocellulose membrane (Figure 3) proved that protein was not fully transferred to the nitrocellulose membrane. Lack of transfer of protein to the nitrocellulose membrane maybe due to the inaccurate rolling of the rod to collect the proteins or maybe the washings were done too many