8 replies to this topic

[crusoe]

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

[bob1]

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.

[doxorubicin]

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.

[crusoe]

bob1, on 02 April 2013 - 12:57 AM, 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 02 April 2013 - 02:06 AM, 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？

[HOYAJM]

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

[crusoe]

HOYAJM, on 02 April 2013 - 07:25 PM, 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.

[mdfenko]

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).

[crusoe]

mdfenko, on 03 April 2013 - 06:29 AM, 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.