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How to estimate the protein expression level from the SDS_PAGE?

expression level protein

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#1 crusoe

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Posted 01 April 2013 - 09:32 PM

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

#2 bob1

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Posted 02 April 2013 - 12:57 AM

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.

#3 doxorubicin

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Posted 02 April 2013 - 02:06 AM

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.

#4 crusoe

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Posted 02 April 2013 - 05:05 PM

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.

#5 crusoe

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Posted 02 April 2013 - 05:11 PM

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?

#6 HOYAJM

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Posted 02 April 2013 - 07:25 PM

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....in/bradford.pdf here is an example

#7 crusoe

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Posted 02 April 2013 - 07:46 PM

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.

#8 mdfenko

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Posted 03 April 2013 - 06:29 AM

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).
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#9 crusoe

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Posted 06 April 2013 - 04:56 PM

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.





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