Protocol Online logo
Top : New Forum Archives (2009-): : Protein Expression and Purification

How to estimate the protein expression level from the SDS_PAGE? - (Apr/01/2013 )

Hi All,
I want to prospect the protein expression level from the little scale cell culture, and then accord the level to scale up.
But I can't estimate well. Does anyone have some suggestion? Or how to estimate it more exactly?
Thanks!
Crusoe

-crusoe-

You can do quantitation on the western blots, but the problem is that it is a very difficult technique to do accurately as exposure times and band intensities are critical for this to work.

-bob1-

Make a standard curve: Purify the protein you are trying to produce, measure the concentration (OD280), and then load known quantities next to your culture sample.

-doxorubicin-

bob1 on Tue Apr 2 08:57:01 2013 said:


You can do quantitation on the western blots, but the problem is that it is a very difficult technique to do accurately as exposure times and band intensities are critical for this to work.


Thank you for your suggestion.

-crusoe-

doxorubicin on Tue Apr 2 10:06:11 2013 said:


Make a standard curve: Purify the protein you are trying to produce, measure the concentration (OD280), and then load known quantities next to your culture sample.


Thanks! Can I load another concentration know protein like BSA to make a comprare?

-crusoe-

BSA is the most common. See if you have a standard set, if not it is not hard to just make your own. To make the standard curve you do a range of BSA concentrations and add bradford reagent and measure the A595. You can then plot these values to generate a line. y = mx + b can then be used to calculate the concentration of your unknown.

http://www.ars.usda.gov/sp2userfiles/place/12650400/glomalin/bradford.pdf here is an example

-HOYAJM-

HOYAJM on Wed Apr 3 03:25:24 2013 said:


BSA is the most common. See if you have a standard set, if not it is not hard to just make your own. To make the standard curve you do a range of BSA concentrations and add bradford reagent and measure the A595. You can then plot these values to generate a line. y = mx + b can then be used to calculate the concentration of your unknown.

http://www.ars.usda..../bradford.pdf here is an example


Thanks.

-crusoe-

although bsa is the most common protein standard, it is not always the best for coomassie unless your protein of interest is similar.

bsa gives readings ~2X those of other gravimetrically prepared proteins (eg bovine gamma globulin).

if you are trying to evaluate a stained gel (rather than a blot) then you can scan the lane and get a rough approximation of each band from their percent of the total (if you know how much you loaded in the lane).

-mdfenko-

mdfenko on Wed Apr 3 14:29:12 2013 said:


although bsa is the most common protein standard, it is not always the best for coomassie unless your protein of interest is similar.

bsa gives readings ~2X those of other gravimetrically prepared proteins (eg bovine gamma globulin).

if you are trying to evaluate a stained gel (rather than a blot) then you can scan the lane and get a rough approximation of each band from their percent of the total (if you know how much you loaded in the lane).

Thank you very much!It's helpful to me.

-crusoe-