SDS-PAGE and specific activity

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faerstd
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SDS-PAGE and specific activity

Post by faerstd » Sun Nov 30, 2008 3:57 pm

Hello,

I purified an enzyme and took samples after each purification step which I loaded on SDS-PAGel and ran SDS-PAGE. I also measured the activities of each sample and calculated the specific activities of them.

To my question, I've heard some time that you can somehow interprete the number of bands of each sample in correlation of the specific activity you calculated...
how can I do that..
first, all I can detect from the bands of each sample is that with each purification step their number is decreasing and that the band corresponding to my enzyme is weakening ..
second, I made a curve for the specific activity and can say that there is a slight increase...

I've already searched on google but all I found was this sentence:
"In analyzing our purification of ß-galactosidase, we will compare the number of protein bands present on an SDS PAGE gel, stained with Coomassie Blue, for each of the different stages in the purification procedure and examine how this relates to the specific activity at each point in the purification procedure." from http://matcmadison.edu/biotech/resource ... chapter_5/
the next chapters do not give any clues how to do it..

how can I correlate the specific activity with the bands on SDS-gel? please help.

thanks in advance.

beautifulmind420
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Post by beautifulmind420 » Tue Dec 09, 2008 9:39 pm

Hi,

I am not sure if this is the right explanation, but here is something to think about...

At each purificiation step, the number of bands should be decreased. In theory, the protein should be pure when subsequential purification do not lead to any increase in specific activity. So as the number of bands decreases, the specific activity should reach to constant. Let me know if this makes sense.

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