I always face problems during protein quantification. In our lab we usually quantify proteins using Bradford and A280. However, we think we have some problems during quantification which is giving lower concentration compared to when we visualized by SDS-PAGE (purified protein in this case).
I would tend to trust the UV absorbance readings more than the Bradford assay or staining in a gel. Bradford reagent - Coomassie G-250 - binding depends on the composition of the protein, in particular the levels of basic residues. If your protein has a significantly different composition to the standard used (e.g. BSA), you will get an erroneous result. If you want to use a colorimetric assay, you could try an alternative such as the BCA assay and see if that helps.
If you need a very accurate concentration, then you could send the sample for amino acid analysis.
If you let us know what are the samples you are estimating protein in, it will help answering. Sometimes in crude extracts there are a lot of interferences. Let me know the biological material from which your sample comes and the status of purity.
sorry..the samples are purified recombinant protein produced from e.coli. well from the sds page i can say it's almost 90% pure. n when compared to BSA with known concentration i estimate my protein should be around ~10ug. however using Bradford, it only gave around ~1ug concentration.
if it's a crude sample, what are the examples of interference that can effect our reading?
I would tend to trust the UV absorbance readings more than the Bradford assay or staining in a gel. Bradford reagent - Coomassie G-250 - binding depends on the composition of the protein, in particular the levels of basic residues. If your protein has a significantly different composition to the standard used (e.g. BSA), you will get an erroneous result. If you want to use a colorimetric assay, you could try an alternative such as the BCA assay and see if that helps.
If you need a very accurate concentration, then you could send the sample for amino acid analysis.
Also the UV asorbance depends on the amount of aromatic aminoacids (Trp, Phe and Tyr, but mostly on Trp). If you have the molar extinction coefficient of your pure protein (from literature, or calculated elsewhere) you can quantify it better than by comparing it with the absorption of BSA or other proteins. I agree with Roger Dodd about the other assay, the BCA (bicinconinic) assay, which is more sensitive and also less dependent on the composition in aminoacids of the protein. However, this assay is sensitive to reducing agents (eg. DTT or beta-mercaptoethanol) so you need to get a rid of them.
About the interferences on Bradford assay, for instance the detergents can cause an overestimation of the protein in the sample.
You might have problems during the quantification protocol (although it is very simple). Can you post a summary of it?
Most probably Matrix Interfearance!
Use same matrix buffer for dillution and for blank.
Is there any observation of any precipitation or degradation while storage or during sample preparation.
Thanks..i do observe some precipitation after one week kept at -20
so it might be this the problem...Did you do the quantification and the sds-page the same day? If so, it is not a problem due to the precipitation bu rather it might be a matrix interference as said by Yamak.
Yes..i did the reading first then sds page on d same day.
Thannks a lot
So, It might be a matrix interference. Try to dissolve the bsa standard in the same buffer of your samples to see if it is ok.
I am having the same issue with total protein quantification in cell lysate from primary smooth muscle cells. I have tried both BCA and Lowry assays and my protein concentration reads as zero while my BSA standards (dissolved in the same lysis buffer) are accurate. However, when I do a Western blot of the samples, there is an adequate amount of protein on the membrane. Has anyone run into this problem? I'm thinking about precipitating the protein samples with TCA in order to remove interfering substances.
It is quite strange that your protein samples do not read despite you used the same lysis buffer also for bsa. In fact, using the same matrix should overcome the issue. Usually with bca the interfering substances are more likely to increase the readings so you overestimate... Or at least i guess. Which is your lysis buffer? Because there is a list of "allowed" substances with their concentrations.
Contamination can interfere with the BCA assay. I suggest a SDS-PAGE control where you compare a standard protein (BSA?) at known concentrations (e.g. 0.01, 0.05, and 0.1mg/ml) with various diluted samples of your protein. This is not a long-term solution to the problem, but it would give you a good idea of your protein concentration. I think precipitating the protein to get rid of contaminants is a good idea.
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