Which method is the best choice to determine protein concentration of amyloid beta peptide (25-35) with a molecular weight of 1060.28 g/mol?
Is Bradford assay incompatible because it responds to proteins with 3000-5000 Da molecular weight?
Hi Mona,
Does your peptide contain an aromatic residue, preferably a tryptophan? If yes, you can estimate the concentration by spectrophotometric technique employing the extinction coefficient of the peptide at 280 nm. You can calculate a rough approximation of the peptide extinction coefficient theoretically at http://web.expasy.org/protparam/ by providing the sequence.
Another method to estimate peptide concentration is based on heat denaturing peptides at high temperature to expose buried hydrophobicity in the presence of alkali and detergent to prevent refolding and employing bicinchoninic acid (BCA) assay to estimate concentration. https://www.ncbi.nlm.nih.gov/pubmed/19539599
I hope my answer is helpful.
Hi Bharath,
Thanks for reply. Here it is the sequence: H-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-OH. No aromatic residues.
You could try this BCA-based peptide assay kit. It even includes a peptide standard:
https://tools.thermofisher.com/content/sfs/manuals/23275_quantpeptide_color_UG.pdf
However, it is important to recognize that your particular peptide may not be well-represented by the standard, which could lead to a systematic error in the measurement.
Measurement of the primary amine content is another possibility. There are a variety of reagents for this, such as OPA (o-phthaldialdehyde), fluorescamine, ninhydrin, and TNBSA.
Here is a TNBSA solution:
https://www.thermofisher.com/order/catalog/product/28997
You could also send a sample out for quantitative amino acid analysis.
With your sequence, the peptide will be invisible to UV-Vis spectrophotometry since it lacks Trp, Tyr and Cys. Hopefully the BCA assay works for you since you have quite a few hydrophobic residues. Best Wishes!
Unfortunately, even nano-drop is bound to fail because, as the name suggests, the spectrophotometer will be able to handle lesser sample in case sample availability is a constraint. However, if the peptide doesn't have any absorbance ( I mean, in the absence of Try, Tyr or Cyst), it will still be invisible to the light. I am not sure whether the peptide bond absorbance at 220 nm can be used for quantification purposes but I am certain the interference will way too high. I saw a similar question on Researchgate which I feel you can refer to gain additional insights.
https://www.researchgate.net/post/How_can_I_calculate_the_concentrations_of_peptides_without_any_aromatic_amino_acids_using_absorption_spectroscopy
Mona, as the author of the paper that Bharath cited, I would encourage you to invest in some synthetic peptide as a standard. The modified BCA assay I published does massively reduce inter-peptide variation, but if you're assaying a pure sample then a pure standard is best for quantification.Add your answer
In case Tryptophan, Tyrosine and Phenylalanine are absent the peptide will not absorb light in the 280nm range. One question that I am curious to know is that whether your peptide folds into any secondary structure or not.
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