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

More documents

Recommendations

Info

6 FIG. 3: The above photograph shows the electrophoresis set-up while it is running. FIG. 4: The top-view of the electrophoresis set-up while it is running.
• The voltage is maintained till the blue line in the gel (formed due to the accumulation of the dye) hits the lower edge of the gel. As a thumb rule the protein is generally behind dye. One shouldn’t leave the process unattended for long at this stage since the proteins can very well run out of the gel into the solution if kept for long. 7 FIG. 5: The faint blue line towards the bottom of the gel indicating the distance through which the dye has run. • After the gel running is complete, the apparantus is dismantled and the glass plates taken out. The two glass plates in every pair will stick to each other very strongly because of surface tension and just trying to pull them apart runs the risk of breaking the fragile glass and also the gel. In general water is lighly run through the upper edge and then softly one can pry them apart using a plastic wedge. • Using the same wedge the gel is lighly detached from the glass plate and this thin film that is formed is transferred to a tray. Now one can either use this gel to do staining with Coomassie blue dye or use it for Western Blotting. The next steps of Coomassie blue staining are relatively simple which are listed below and Western Blotting is a longer sequence of steps which was unsuccessfully tried and shall not be explained here. The steps of staining and destaining are as follows, • On the gels which are transferred into the tray, Coomassie Blue dye solution is poured and the set up is put on a rocker for overnight staining. This is staining is generally done for about 10 − 12hours and during this time the oscillations of the rocker ensure that the dye keeps flowing across the gel uniformly without clustering anywhere and hence stains the gel uniformly. • After the staining is over the remaining staining solution is recovered for reuse. • The destaining solution is is then pourd in and set on a rocker. As the stain dissolves into the destaining solution the solution will keep becoming blue and then one decants out the solution and pours in fresh solution. This change has to be done almost every 10 − 20 minutes for about 5hours. • During this destaining process care must be taken not to overdo the process since it can eventually start dissolving out the dye stuck to the protein bands also. • Initially the gel looks uniformly blue after about 2 rounds of destaining the protein bands become visible in contrast with the lighter background.
Page 1 and 2: SDS-PAGE Electrophoresis Anirbit De
Page 3 and 4: 3 To make Staining Solution one mix
Page 5: 5 D. Making BSA dilution solution T
Page 9 and 10: 9 FIG. 7: The basic idea is of two
Page 11 and 12: 11 H. Image analysis on the gel Aft
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

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?