SDS-PAGE

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biology_06er
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SDS-PAGE

Post by biology_06er » Mon Mar 23, 2009 3:58 am

Hi there,

can someone please explain how to do the following:
ok, so what we did in our lab was that at the end of the practical we loaded 4 samples using SDS-PAGE, they were

sample 1-purified GST
sample 2-E.coli lysate from RAP-pGEX-2T cells before IPTG induction
sample 3-E.coli lysate from RAP-pGEX-2T cells after IPTG induction
sample 4-our purified GST-RAP protein sample
and we also loaded a molecular mass protein ladder.

in our book we have a picture of a ladder starting from 10kD up till 250kD and next to it it has a bunch of proteins such as albumin from bovie 66000Da and Trypsin Inhibitor from soybean 20100Da.

It then goes on to say make a plot of log (molecular weight) against relative mobility for each standard protein. use the measured mobilities of the proteins in your SDS-PAGE gel, not from the picture given in our book.

soo...looking through the int/text book it says relative mobility is calculated by the distance moved by a protein standard divided by the distance moved by bromophenol blue (a marker of the electrophoretic front).

how do i graph this: on the y-axis do i just log the molecular weights (from 10kd to 250kd) and then plot it against what??...im confused as to why albumin and other protein weights are given.
do i measure the distance that my gel shows up: ie the purified gel has moved 2.6cm?
any help will be greeeatly appreciated!!

Cheers,
b_06er

blcr11
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Re: SDS-PAGE

Post by blcr11 » Mon Mar 23, 2009 10:37 am

Each standard protein will be an (x,y) point on the graph where x = relative mobility and y = log(MW). Once you plot all the standard proteins, you should be able to draw a reasonable line through the points (or do a regression). This is your calibration line. Now find the relative mobility of the unknown protein on the x-axis. Draw (or imagine) a vertical line extending up to the calibration line. From the intersection of the vertical line with the calibration line draw (or imagine) a horizontal line extending to the y-axis. Read off the value at the intersection of the horizontal line with the y-axis. It is your estimate of the log(MW) of the unknown protein. Take the antilog of that number and you have an estimate of the MW of that protein. If you did a regression to find your calibration line, you could just plug in the value of the relative mobility of the unknown as a value of "x" and ask the calculator to find the "y". This will give you an estimate for the log(MW) without having to plot anything. This is the way you use calibration curves in general.

ash89
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Re: SDS-PAGE

Post by ash89 » Mon Mar 29, 2010 4:54 pm

what if a protein data sheet provided only had a range of relative molecular mass to find out what protien is present? how would you go from knowing the MW to being able to work this out?

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