A report on an experiment I did of doing electrophoresis with proteins

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• The Glycine provides the ion with the slowest mobility which helps in stacking the proteins during the transport in the presence of the electric field. • The Chloride ions provide the background conducting medium and is the “leading ion” being the ion around with the highest mobility. Hence the proteins being of intermediate mobility get stacked between the chloride and the glycine ions. • Bromophenol Blue is employed tracking dye mixed in the protein solution, because it is viable in alkali and neutral pH, it is a small molecule, it is negatively charged above pH 4.6 and hence moves towards the anode. Being a small molecule it moves ahead of most proteins and nucleic acids. As it reaches the anodic end of the electrophoresis medium electrophoresis is stopped. It can bind with proteins weakly and give blue colour. 10 FIG. 9: Structure of Bromophenol Blue • Coomassie Blue R-250 is an anionic dye used in the staining solution.This binds with proteins non-specifically. As SDS is also anionic, it may interfere with staining process. Therefore large volume of staining solution is recommended for use. FIG. 10: Structure of Coomassie Blue
11 H. Image analysis on the gel After the gel has been destained the objective is to quantify the mass of the proteins from the instensity of the dye that is stuck to it. An exact quantification has its own complications and I shall explain how far one can go in this goal. Basically the idea is to see how the intensity of the gel varies as a function of the protein mass and this requires a way of quantifying the intensity. For this the gel is photographed and the sum of the pixel values is taken as a proxy for the intensity since one knows that bighter points in the picture have higher pixel values. Then all analysis can be done using this set of numbers as obtained by the following process of “digitizing” the gel. The exact steps in the process are as follows, • The last destaining solution is not transferred out of the tray. • A pair of soft smooth transparent plastic sheets is cut out of the size little bigger than the gel and these are joint at one edge. Usually this is made by cutting out from the edge of plastic packets. • Keeping the gel dipped inside the solution a hard plastic is slid underneath it and the gel is lifted out of the solution on it. This method ensures that no air bubbles are trapped between the gel and the plastic. • Then gently the gel is slipped on to one of the pair of soft palstic sheets from the hard plastic by sliding it onto it keeping the leading edge always in touch with a thin film of water. This ensures that no air bubbles get trapped in this stage. • The second plastic film is covered over it in a similar way ensuring that no air bubbles are trapped. FIG. 11: The gel as it looks just after taking out of the destaining process • This setup of the gel trapped between pairs of soft tranparent plastic sheets is gently put on a scanner and the image is taken at a resolution of 600dpi. • From the image the relevant part is cut out (generally a thin rectangular strip corresponding to the dilution series) and rotated if necessary. FIG. 12: The strip of the scanned image of the gel along the dilution series
Page 1 and 2: SDS-PAGE Electrophoresis Anirbit De
Page 3 and 4: 3 To make Staining Solution one mix
Page 5 and 6: 5 D. Making BSA dilution solution T
Page 7 and 8: • The voltage is maintained till
Page 9: 9 FIG. 7: The basic idea is of two
Page 13 and 14: 13 FIG. 14: A pot of the points (1
Page 15 and 16: 15 FIG. 18: The above graph shows t
Page 17 and 18: But for a fixed protein if m 0 and
Page 19 and 20: 19 E l ∑i [u i]µ iu z iu = E u

A report on an experiment I did of doing electrophoresis with proteins

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