I received 0.5mg of amyloid beta from a company and dissolved it into 0.5ml of 1mM NaOH to make a 1mg/ml solution of amyloid beta 1-42. I then aliquoted the peptide and performed a BCA on the peptide. I am measuring 521ug/ml. A representative from the company informed me that doing a BCA on amyloid beta would not give you an accurate readout of the protein concentration. Does anyone know if this is true or has anyone done BCA assays on known amounts of amyloid beta? Any information would be helpful!
Thanks,
Mandi
typically, in order to get an absolute quantification of protein concentration you meed to use the protein of interest in order to construct the standard curve. In your case this is not feasible though, so in this case I would move forward considering that protein concentration is 1mg/mL
Each protein behaves a little differently in colorimetric assays, so using a different protein as the standard (BSA?) introduces a systematic error. The error can be large if the amino acid compositions of the protein of interest and the standard protein are quite different. I agree with Paschalis - since you know the weight of the protein sample (0.5 mg) and the volume you dissolved it in (0.5 ml), you should take that solution to be 1 mg/ml. In the future, if you want to use the colorimetric assay on an Abeta sample of unknown concentration with the same standard as before, you can correct the measurement for the known bias by multiplying by 1/0.52.
Thank you Paschalis and Adam for the feedback! It would be very strange and unlikely that a company would be sending half the amount of product that they intended. There must be some error in this.
Hi Mandi,
I'm quite late to answer. anyway here is the corrsponding literature:
http://www.ncbi.nlm.nih.gov/pubmed/20539293
there is a section where the authors compared different methods for determining the concentration of abeta. It apears that you have to multiply your BCA result based on BSA by some factor to get the real Abeta quantity.
According to this protocoll:
http://www.amidebio.com/files/Data/Abeta_Quantitation_Protocol.pdf
this correction factor is 1.51.
Hi Christian,
Sorry for my delayed response to this. It's really nice to have a publication that has analyzed this. Thank you for sharing!!
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