I am working with a construct containing only the C-terminal region of my POI. It is 42 residues long, and contains no aromatic residues (but does contain a number of basic amino acids). Following SDS PAGE and Coomassie staining there is a large band visible on the gel. However on Bradford assay the concentration is determined to be very low (130 µg/ml). The concentration using BCA assay is a little higher (200 µg/ml) but I still suspect this is an underestimation. I have a number of questions.
1) Why is there such a difference between the appearance on SDS PAGE and Bradford, since these both use Coomassie based dyes and therefore are not due to differences in amino acid composition?
2) Since I do appear to get good staining on SDS PAGE, perhaps I could estimate the concentration by running BSA standards on the gel and analysing by densitometry. However I am unsure whether it is relevant to compare intensity of staining of a small peptide (6 kDa) and BSA on SDS PAGE in this way? Is molecular weight relevant here?
3) Can anyone recommend any alternative methods?
Any advice much appreciated!
Your case is indeed a bit tricky since 42 residues would mostly fail with composition-based dye-aided concentration estimators. BSA-based assays that depend on the amino-acid composition of your peptide/protein are plagued by so many unpredictable variables that you may often times get these aberrant anomalies .Expasy provides this utility called protparam ( for protein parameters) at http://web.expasy.org/protparam/.
Try keying in your sequence and get the theoretically computed extinction coefficient from the website. Do a wavelength scan of your protein and use the extinction coefficient to compute back your concentration. Hopefully you get a far better readout than these dye based concentration estimations are giving. Do let me know how it went. Best
1) There could be a large number of reasons for the differences in the "appearance" of the dye in the Bradford assay as compared to the SDS gel - the biggest one being the presence of SDS.
2) Gel based densitometry would be my last hope, if all else fails (I guess thats a personal bias). I prefer the BCA assay which is not as dependent on amino acid composition as the Bradford (unless you have a bunch of cys/tyr/trp residues).
3) Alternative method - label the terminal end of your peptide with a reactive dye. For example, if you use a primary amine reactive dye - it will label your N terminal residue as well as any primary amines on side chains. Since you know the sequence, you can calculate the number of dye molecules expected to react per peptide chain. Quantifying the reacted dye will give you a quantification of your peptide.
Bottom-line: unless you have a lot of cystine residues (you already said you don't have any aromatic ones), I think you can trust the BCA assay (it is certainly much less involved than labeling). It seems like you are doubting the BCA because you are comparing the stain on the SDS-PAGE - which is not really comparable.
Good luck!
Hi Martin,
You can try OPA method (O-Phthalaldehyde).. Its a fluorometric assay that can be used to determine the concentration of peptides..
Best of Luck..
Hi Martin,
the answer to your question depends on whether you will need an absolute quantification or a relative one. For relative estimation of the concentration, we use RP-HPLC in Acetonitrile/water. You might prepare serial dilutions of your peptide and measure the area under the peak for construction of a calibration curve.
For absolute quantification Edman sequencing is a practicable method, because you will get absolute masses of the different amino acids.
Much easier is the following procedure:
You collect the peak of the RP-HPLC, get rid of the solvent for instance by evaporation and estimate the weight of the dry substance using a microbalance.
Hope that helps,
Juergen
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