Hello everyone,
I wish to measure the total protein concentration of some samples but my standards are always bended rather than giving a nice straight line.
I first tried to make the standards with BSA but also tried Ovalbumine. Here I though that maybe my Bicinchoninic acid has went out so I bought a BCA kit but I keep having the same bended standard curve (see image)...
Has anyone any idea of what is going on? What can I try to fix that?
Thank you for your help,
Best regards,
The standard curve bends because there is not enough of the detection reagent to bind to/react with the higher concentration of proteins. Beyond that, there is a linear phase (500-2000), which may be due to precipitation of the protein scattering light.
The usable part of the standard curve is the linear part in the lower concentration range (0-125).
Thank you Adam B Shapiro
So if I increase the amount of reagent should I have a more linear curve?
I am surprised because I followed the kit protocol in which they say that I should obtain an "nearly" linear curve over the range of 20-2000ug/mL (see picture)
Dear Lora,
I use the same BCA assay from Thermo (Pierce™ BCA Protein Assay Kit).
https://www.thermofisher.com/order/catalog/product/de/en/23225
if you follow the user manual properly (BSA as standard, correct dilutions in a suitable diluent, duplicates, fresh working solution, incubation for 30 min @ 37°C, quick measurement in a photometer at the correct wavelength of 562 nm), you should get a curve like the one in Fig. 1. I prefer the measurement in cuvettes.
https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FMAN0011430_Pierce_BCA_Protein_Asy_UG.pdf
There is a Tech tip on the Thermo website dealing with the use of the assay standard curve.
https://assets.thermofisher.com/TFS-Assets%2FLSG%2FApplication-Notes%2FTR0057-Read-std-curves.pdf
Good luck
Michael
Thank you Michael Kohlstedt
I did follow each step of the protocol but I used it in microplates, not in cuvettes.
The only thing I wasn't sure is what is "a suitable diluent"?
I used PBS as it was the solution I used to extract my cell lysate but maybe that is not suitable?
Thank you very much for your help
PBS should be fine.
https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0068-Protein-assay-compatibility.pdf
Best
Michael
You can use water as the diluent. PBS should also be OK.
Since you are working in microplates, you should be using the microplate method, as detailed here (starting on page 3):
https://web.stanford.edu/class/chem184/materials/BCAmanual.pdf
Thank you for sharing your standard Michael Kohlstedt
Adam B Shapiro yes, I used the microplate method, using the 1:8 ratio they advise.
I will give it another go this week
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