5 replies to this topic

[Riccardo Guidi]

I want to share my case to get some of you feedbacks.
I recently found out a great discrepancy in the total protein concentration assay I run between two very well established systems: Bradford and Lowry.
Bradford comes from Sigma (http://tinyurl.com/d8y969f), Lowry is the Bio-Rad DC kit (http://tinyurl.com/c7uszdh). This forum has discussed these two system extensively, but never compared the two. Agains my expectation, for one protein preparation the two systems gives very different results, as indicated in the picture.



from 0.1 to 3.2 I have BSA standards (g/L) in 20% glycerol/PBS. The bottom line is my sample in same buffer as standards. Each measurement comes in duplicate.
As a note, I run my SDS-PAGE + Wester Blot of the samples and no Ponceau S appeared in the line of the samples (other samples were fine). Antibody detection also failed to reveal any band were I expected.
As a result, I now rather say that of the two option, Bradford seems to be true.

How can I have such dramatic difference?
Anyone experience something similar?

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[mdfenko]

showing a picture of the assay doesn't tell us the result.

are you saying that the bradford shows a lower concentration of your sample than the lowry? if so, then the probable reason is the standard you selected (bsa).

color generation of the bradford assay is ~2x with bsa than with bgg. biorad recommends bgg be used as standard when determining most proteins (see the section on selection of standard).

Edited by mdfenko, 10 December 2012 - 08:12 AM.

[Riccardo Guidi]

Sorry if my explanation was not clear. In Lowry, the sample intensity is higher then the latest point in my standard curve (>3.2 g/l BSA). In Bradford, the sample intensity is less then 0.1 standard. I agree with you I should have run the colorimetric analysis in this, but I the difference was so clear that I skip it.
I can try BGG if I have it in house. Still, if bradford is about 2-fold more intense with BSA, doesn't tell why I have at least 10-fold difference :-/

[mdfenko]

the primary structure of the protein of interest may be at fault.

a third method should resolve the issue of which assay method is correct.

as you said, ponceau staining points to the bradford being in the ballpark (may be more accurate with bgg standard).

do you have access to any other protein assay methods (maybe something like fluorescamine or direct detect)?

[Riccardo Guidi]

I have a NanoDrop which potentially could give me the total amount of purified proteins based on the UV absorption. But again, it's recommended for pure proteins. I'll ask around some BGG (I dont have it) and try with that standards.

thank for helping

[memari]

One of them is sensitive to detergent and other interfering agents.
Check it.

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Babak Memari