How to quantify the protein amount for serum samples ? - (Dec/12/2011 )
I have to do a western blot test to serum samples.
How can i quantify the amount of the total proteins to ensure equal loading ?
I'm not sure if a Bradford protein quantification test (BCA kit) will work well because that there is a color difference between the samples that could effect the test results.
Blood plasma samples were produced from blood samples using 0.5% Heparin solution and centrifugation.
first, bradford and bca are not the same.
since you have interfering color you will not be able to use methods in which you determine absorbance. you may be able to use the biuret method but it is (a lot) less sensitive than the two you mentioned (which may be okay for serum and plasma) but may also be affected by the intrinsic color of your samples.
you may be able to use a method that uses fluorescence, it may not be quenched by the interfering color.
one fluorescence method for protein determination uses fluorescamine. it is very sensitive so you will have to use very small amounts of serum (or plasma? you mention both in your post).
here is a link to a comparison table of protein assays (absorbance and fluorescence based):
invitrogen/molecular probes table 9.1
Serum total protein estimated by such macro protein techniques as the Biuret method whidely used. It’s adequate, despite the known limits, to the requisite of diagnostic acceptability. In my practice employment a kit two reagents home made with stater as following suitable :
Preliminary reagent : NaOH 5.0 M : dissolve 200 g of NaOH in freshly distilled water and dilute to one liter. Store in a tightly closed polyethylene bottle. Use a new bottle of NaOH, to assure that it is dry and free of Na2CO3.
Blank biuret reagent (R1) : dissolve 4.8 g <17 mM> of K-Na-tartrate in dH2O. Add 20 ml <100 mM> of 5.0 M NaOH and dilute to 1 liter with water. Store in a polyethylene bottle. Stable 3 years.
Biuret reagent (R2) x 100 ml : dissolve 0.75 g (± 0.01 g) <30 mM> of CuSO4•5H2O in 50 ml of freshly distilled water. Add 2.26 g (± 0.01 g) <80 mM> of K-Na-tartrate and 1.245 g (75 mM) of KI. After the solution is complete add 10 ml (20 g/L – 500 mM) of 5.0 M NaOH and dilute to 100 ml with dH2O. Store in a tightly closed polyethylene bottle. Stable 3 years.
Procedure : dispense in 3 label tubes (Blanck, Standard, and smple) 1 ml of R1 and 20 μl standard or sample respectively. Mix, read absorbance A1 against the reagent blank after 1-5 min. at 20-25°C/37°C, at 546 nm than add in all tubes 250 μl R2. Mix, incubate for 5 min. at 20-25°C/37°C and read absorbance A2 against the reagent blank within 60 min. at 540 nm. ΔA = (A2 – A1) sample or std/cal