How to estimate the protein expression level from the SDS_PAGE? - (Apr/02/2013 )
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
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.
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.
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.
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?
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 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.
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 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.