Determination of Protein Concentration

Several methods are commonly used for determination of protein concentration. Measurement of the UV
absorbance at 280 nm is most useful for pure protein solutions. Bradford and BCA assay methods are routinely
used during protein purification and screening. Novexin has developed a novel assay formulation, Bradford
ULTRA, with the features of the standard Bradford assay and the added advantage of greater tolerance of
detergents.

Table 1: Comparison of Bradford, Bradford ULTRA and BCA protein assay methods.

PROTEIN DETERMINATION USING ABSORBANCE AT 280 nm
(Ref: Pace, C.N. et al, (1995) Protein Sci,. 4, p2411.)
If a protein sequence is known, the theoretical extinction co-efficient at 280 nm, ε280nm, can be estimated using the
equation
ε280nm (M-1cm-1) = (#Trp)(5500)+ (#Tyr)(1490)+(#Cys)(125)
    • Warm up the UV lamp (about 15 min).
    • Zero spectrophotometer to buffer at 280 nm in a quartz cuvette.
    • Measure the absorbance of protein solution at 280 nm in a quartz cuvette.
    • Protein concentration is calculated as:
            [Protein] (mg/mL) = A280nm /(ε280nm x (cuvette path length in cm))

Note! An economical and disposable alternative to quartz cuvettes are cuvettes made from optical grade polymethyl
methacrylate (PMMA), eg Fisher Scientific, Disposable UV Cuvettes, #CXA-205 series.
NVoy technology is a quantum leap in protein processing, production and analysis. It uses
proprietary NV polymers to enhance protein solubility and stability through the formation of
multi-point reversible complexes with proteins without altering their structure.

PROTEIN DETERMINATION USING THE BRADFORD ASSAY
(Ref. Bradford, M. M. (1976) Anal. Biochem. 72, p248.)
The Coomassie Brilliant Blue G-250 dye binds selectively to positively charged residues (lysine, arginine and
histidine) and aromatic residues, and this binding is accompanied by a shift in absorbance maximum from 465 nm
to 595 nm. The assay is fast, inexpensive and sensitive, and tolerates a wide range of buffers. The Bradford assay
is, however, protein dependant, non-linear and detergent incompatible. Novexin has developed a detergent
compatible assay solution, Bradford ULTRA, which removes the requirement for detergent-solubilised protein to be
precipitated before use.
Note! The Coomassie dye binds to quartz, so it is advisable to use glass or plastic cuvettes.

PROTOCOL for Bradford ULTRA
    • Mix the Bradford ULTRA Reagent solution immediately before use by gently inverting the bottle several times
        (Do not shake the bottle to mix the solution). Remove the amount of reagent needed and equilibrate it to room
        temperature before use.

    • Make a dilution series of the chosen model protein in the range:
        0.1 mg/ml – 1.5 mg/ml (high protein range) OR
        1 μg/ml – 25 μg/ml (low protein range)
    • Mix the samples, standards and a blank (buffer, no protein) with Bradford ULTRA reagent in a microtiter plate:
            o For 0.1 mg/ml – 1.5 mg/ml protein (high range): 20 μl sample + 300 μl Bradford ULTRA reagent.
            o For 1 μg/ml – 25 μg/ml protein (low range): 150 μl sample + 150 μl Bradford ULTRA reagent
    • Read absorbance immediately at 595 nm.
    • Subtract the average 595 nm measurement for the blank from the 595 nm measurements of all other
        individual standards and unknown samples. Plot the average blank-corrected 595 nm measurement for each
        standard vs. concentration. Use the slope of this standard curve to estimate the protein concentration of the
        unknown samples.

NVoy technology is a quantum leap in protein processing, production and analysis. It uses
proprietary NV polymers to enhance protein solubility and stability through the formation of
multi-point reversible complexes with proteins without altering their structure.
 

PROTEIN DETERMINATION USING THE BCA ASSAY

(Ref. P.K. Smith et al. (1985) Anal. Biochem. 150, p76.; K. J. Wiechelman et al. (1988) Anal. Biochem. 175, p231.)
This is a two-step assay, in which Cu2+ is first reduced to Cu1+ forming a complex with protein amide bonds (Biuret
reaction). Secondly, bicinchoninic acid (BCA) forms a purple complex with Cu1+ which is detectable at 562nm. The
assay is sensitive and linear but is relatively slow unless heated.

PROTOCOL for BCA Assay

Reagent A:                        • 1 g bicinchoninate (BCA), 2 g sodium carbonate, 0.16 g sodium tartrate, 0.4 g NaOH,
                                            0.95 g sodium bicarbonate.
                                         • Mix reagents in 80 ml distilled water, adjust pH to 11.25 with 8 M NaOH, and make
                                            solution up to 100 ml.
 

Reagent B:                         • 4% CuSO4.5H2O (4 g in 10 ml distilled water).

Working solution (WS):       • Mix 50 volumes of Reagent A with 1 volume of Reagent B.
                                          • This green solution is stable for 1 week.

• Make a dilution series of the chosen model protein (eg BSA, IgG) as 100 μL samples containing 0 - 100 μg
    protein
• Add 2 ml of WS to each 100 μL sample or standard.
• Seal samples and incubate at 60 oC for 15 minutes (or 37 oC for 30 minutes).
• Cool samples to room temperature and measure the absorbance at 562 nm.
• Subtract the average 562 nm blank from the 562 nm measurements of all other standards and samples. Plot the
    average, blank corrected measurement for each standard vs. concentration. Use the slope of this standard curve
    to estimate the protein concentration of the unknown samples.