Protein quantification with BSA?

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Protein quantification with BSA?

Postby celvas » Nov 14 2007 12:37 pm

To much people quantify its protein samples with a BSA standard curve.
But, this way to determine the concentration is the same as reading the sample OD and then apply the BSA extinction coefficient (which would be faster), isn't it?
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Postby relaxin » Nov 14 2007 1:58 pm

Different proteins have different number of aromatic amino acid residues, and hence different extinction coefficient. Your method may give you results that are way off. Other colorimetric (dye binding) methods do not depend solely on the number of aromatic amino acid residues.
Not affiliated with any company. Mention of a specifc product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
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Postby Protein Wizard » Nov 15 2007 2:14 am

We have routinely used OD280 (A280) to measure protein concentration. You can get extinction coefficient of a known protein using many sequence analysis programs or assume 1 OD280 = 1 mg/ml for mixtures. In most cases, they come very close to Bradford assay with BSA standard for purified proteins. This method has limitation at early stage of purification. Make sure OD260 is lower.
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Postby kamome » Feb 15 2008 2:52 am

Protein Wizard wrote:Make sure OD260 is lower.


Hi. May I ask why it should be lower? I was in the understanding that different proteins can have different OD280/OD260 ratio, and that this can actually be used to tell them appart in a chromatogram (if the difference is big enough of course).
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Postby jk » Feb 15 2008 6:16 am

If your Abs260 is higher than Abs280, then you have not only protein but also DNA in your sample.
With Bradford assay you can measure _only_ protein, even when you have DNA.
Once you have pure protein sample, you can use Abs280 and Bradford assay to compare the two.
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Postby kamome » Feb 17 2008 7:59 am

OK, so "different ratio" does not mean >1 for one and <1 for the other. Thank you for clearing that up.

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Postby stprotein » Feb 28 2008 9:34 am

As other posters have noted, A280 is a reliable method for quantifying the protein concentration of a pure protein sample with known sequence. It is also true that the A280 should be greater than the A260. I can hunt down a reference if someone needs it, but generally a ratio of A280 to A260 in the area of 1.7 means that there is little/no DNA in your protein sample. DNA absorbs more at 260 than at 280 and in fact A260/A280 ratios are often used to determine DNA purity.
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