I am doing western blot experiment and in protein assay, with BCA reagent, I could not achieve my required loading protein amount. I could achieve only 4 or 5 microgram per microliter. But I could achieve even 50 microgram per microliter with Bio-rad protein assay. It is really weird. So, I used Bio-rad protein assay. However, after ponceau staining, I did not see equal protein bands. Can you please check my protein assay whether it is right or not. I have attached file below.
Thanks in advance.
Dear Khin
in which buffer is it your proteins?
it is contain reducing agent?
did you performed a blank adding the same buffer to the bca?
- Looking at Bio-rad.xlsx I noticed that your calibration curve is in mg/ml, but the calculated concentration of your samples is in µM. That doesn't match. Also, the absorbance of your samples is higher than the maximal absorbance of your standard curve. Such extrapolation is imprecise, as the absorbance curve will start to flatten eventually, Bradford's assay around 0.4 or so. Indeed, a parabola will fit your standard data better than a straight line.
- The same problem also exists with BCA.xlsx.
- Apart perhaps from Kjedahl's method, there is no absolute protein determination, only relative (as much colour as x mg/ml of standard y). Since different proteins have different amino acid compositions and hence different colour yield, your results will always depend on which method you use for which protein. Bradfords assay will give higher results with hydrophobic proteins, Lowry and BCA with proteins containing many reducing aa, UV absorption with proteins with a lot of aromatic aa and so on.
- BSA has hydrophobic pockets (for hormone binding), which in Bradford's assay bind more dye than other proteins. The colour yield hence is artificially high, use IgG as standard protein instead.
Dear Sir all,
I searched for apoptosis signaling protein like PARP. I used lysis buffer containing reducing agent in the second time extract. For calculation, I switched the value directly from mg/ml to microgram/microliter in Bio-rad standard curve and from microgram/ml to ng/microliter in BCA standard curve. I will also try with the parabloa curve. I got results and I attached file of this experiment result. I used Bio-rad reagent. Thanks a lot.
Dear Khin
i think that rhe presence of the reducing agent is not compatibile
with bca and it can determine a huge error in your protein concentration estimation.
The BCA assay primarily relies on two reactions. First, the peptide bonds in protein reduce Cu2+ ions from the copper(II) sulfate to Cu+ (a temperature dependent reaction). The amount of Cu2+ reduced is proportional to the amount of protein present in the solution. Next, two molecules of bicinchoninic acid chelate with each Cu+ ion, forming a purple-colored complex that strongly absorbs light at a wavelength of 562 nm.
if you have a reducing agent, as dtt, it will directly reduce cu(ii) to Cu(i) and it will be detected from bca. this means that if you do not subctract the value of the blank obtined with the buffer you can overstimate a lot the protein amount but Also if you performing this subctraction you evaluation will be not accurate for 2 main reason:
1) high backgruond can determine saturationbin the sprectrofotometric read ( absorbances at 562>1) that are out of the instruments linearity.
2) if reducing agent react with ak the cu(ii) present in the bca the reagent can become limiting and not enough to react also with the protein.
So in this case i think that yuo can greatly understimate yiur sample.
Bradford is not sensitive to reducing agent and i think that can be more reilable in this condiction.
an other possible alternative is nanoorange from thermo (cod n6666) is it quite expensive but nice abd fast and it is not affected by reducing agents.
good luck
Manuele
Manuele Martinelli is right, BCA measures the Cu+ produced from Cu2+ through reduction by peptide ponds. Any redox agent will interfere.
Dear Sir,
Thank you for your answers. At the first time, I used lysis buffer containing HEPES 50mM, EDTA 5mM, NaCl 50mM, 1% Triton X-100 20% and 1%NP-40 20% with protease and phosphatase inhibitors and I sonicated.
For the second time, I used lysis buffer containing Tris HCl, Triton X-100, SDS, reducing agent beta-mercaptoethanol 0.4% with protease and phosphatase inhibitors and I sonicated.
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