I usually use the Bradford Assay, but have had problems (high values... I used 1: 100 dilution, Should I dilute it more?). These serum samples are from rats that have consumed CD or HFD, and a polyphenolic extract supplementation.
What do you recommend me to do?
Biuret is also a very good and reliable assay for protein determination
The concentration of protein in serum is very high, mostly due to albumin and immunoglobulin. If your samples are exceeding the standard curve, dilute them more.
The Bradford method is adequate but you need dilute more the samples, it would be good to test with several dilutions to optimize the assay.
Bradford assay is notorious for the nonlinearity of the commercial assay reagent (>0.8 absorbance values) and laboratory-prepared reagent (>0.35 absorbance values). Absorbance readings above these limit usually need the sample to be diluted further so that the absorbances fall in the linear range, making the Bradford assay not easy to work with. Switch to BCA assay.
I also think that the BCA assay is more reliable, as Yunus Shukor mentioned already. However, be aware that all these assays are semiquantitative in a strict sense. It very depends on the exact question you want to answer.
According to my experience Biuret works best for serum samples. Mostly you won't need any dilution, except for very high concentration. It is based on principle that substances which contain two –CONH2 groups joined together directly or through a single carbon or nitrogen atom, and those which contain two or more peptide links, gives a blue to purple colored compound with alkaline copper solutions. One copper atom complexes with four molecules of Biuret, the linkages being central nitrogen atom. The reaction is thus given by protein.
CO -NH -CONH2 Biuret complex
Standard curve by: Bovine serum albumin
Biuret Reagent: Sodium hydroxide 8.8g, Potassium iodide 5.0 g, Sodium potassium tartarate 9.0 g, Copper sulphate (CuSO4.5H2O) 3.0 g, dissolve and final volume to 1000 ml in DDW.
Procedure: Mix 0.1 ml of serum with 2.9 ml of DDW and add 5.0 ml Biuret reagent. Vortex the tubes and incubate at 37°C for 10 minutes. Then read the absorbance at 540 nm spectrophotometrically. Calculate concentration in g/dl.
I've not used it for serum samples, but I've always found the BCA assay to be very reliable in measuring total protein concentration. I highly recommend the Rapid Gold BCA assay kit that Thermo-Fisher sell, as it gives results in about 10 minutes compared to an hour with a traditional BCA assay and works excellently in a microplate format as it only uses 20 ul of sample (or even 10 ul if you are really limited). The BCA assay is only linear over about 20 - 2000 ug/ml, therefore prepare several serially diluted samples if you are completely unsure of the concentration.
Thank you all! Finally, I ran Bradford Assay with BSA, but I increased the serum dilutions. Thank you very much again.
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