Purification of proteins from human tissue - SDS_PAGE (Aug/16/2005 )

I plan running SDS-PAGE to get the protein profile of human tissues. Which kit is best for this purpose and how can i measure the concentration of a mixture of different proteins? I can measure the protein concentration when it is just a single kind of protein in the mixture, but how about when it is a mixture of different porteins?

Thanks for any helps and ideas!

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hiya,

i'm purifying microtubule and it's MAPS (microtubule associating protein) from Aplysia.
I use Bio-Rad kit for SDS PAGE and it works well. Also, I stain the total protein for each step of purification (i use centrifugation to pull down the proteins) using silver staining protocol. This way I can visualise the each purification step.

Next, I use BCA assay for assaying concentration, this assay can detect mixture of proteins. Well most all of known proteins of course . This one works quite well with low deviations between duplicates. However, bear in mind that as soon as the concentration decreases to a very low range, the sensitivity of this method also decreases (ie, the deviation between duplicates are more erratic!!!).

There are regular procedure and also enhanced procedure for this assay. Regular should works just fine with sample with relatively high concentration. for low concentration, you might wanna use enhanced procedure.

You could also try to use internal standard instead of enhanced procedure (add known amount of standard into the lower concentration so that we can raise the total concentration to that of good working range!).

remember to blank with the correct solvent (ie, use the vuffer as the blank to minimise matrix error).

Can also use standard addition-add known amount of standards into all of the fractions, also to raise the total concentration to a good working range. it's almost similiar to using internal standard in fact

hope you understand. sorry for bombarding with details. Good luck and don't forget any feedbacks!

Cheers!

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