Description
The Bicinchoninic Acid (BCA) protein assay for the quantification of proteins is a highly sensitive colorimetric assay that is compatible with detergent solubilized protein solutions. Similar to the Lowry method, the principle is based on the Biuret reaction in which Cu2+-protein complexes are formed under alkaline condition. As proteins react as reducing agents, Cu2+ is reduced to Cu+, which is subsequently chelated by two BCA molecules, forming a purple complex that exhibits strong absorption at 562 nm. As the amount of Cu+ is proportional to the amount of protein, measuring absorption can be used to quantify protein concentration by comparing with the absorption of protein solutions of known concentrations. In comparison with the Lowry method, the BCA method is not only easier and faster, but also suffers less interference from non-ionic detergents and salts. It is insensitive to the presence of detergents such as SDS (1%) and Triton X-100.
GRiSP´s BCA Quantification Kit (Broad Range) is formulated for the quantification of total protein concentration ranging from 20-2000µg/ml. It is suitable for usage with standard spectrophotometers as well as with microplate readers. The whole procedure can be carried out in less than an hour.
Features
Compatibility
GRiSP´s BCA Quantification Kit (Broad Range) is compatible with the following detergents up to the given concentrations: 5% Brij®-35, 1% Brij®-56, 1% Brij®-58, 5% CHAPS, 5% CHAPSO, 5% Deoxycholic acid, 5% Nonidet P-40 (NP-40), 5% Octyl ß-glucoside, 5% Octyl ß-thioglucopyranoside, 5% SDS, 1% Span® 20, 5% Triton® X-100, 1% Triton® X-114, 1% Triton® X-305, 1% Triton® X-405, 5% Tween®-20, 5% Tween®-60, 5% Tween®-80 and 1% Zwittergent® 3-14.
It is also not interfered by the following reducing and thiol containing agents up to the given concentrations: 10% N-acetylglucosamine (in PBS), 1mM DTE, 1mM DTT, 10mM Glucose, 0.01 ß-mercaptoethanol, 3M Potassium thiocyanate and 0.01% Thimerosal. Moreover, the presence of chelating agents EDTA up to 10mM and Sodium Citrate (up to 200mM) have no negative effect on the outcome of the assay.
In general, chelating reagents or reagents that change the pH or reduce copper are known to interfere with the assay. These include ascorbic acid, cathecholamines, creatinine, cysteine, EGTA, glycerol (impure), hydrogen peroxide, hydrazides, iron, lipids, melibiose, phenol red, sucrose (impure), tryptophan, tyrosine and uric acid.
Protocols
Protocol for Spectrophotometer
- Pipet 100µl of each standard and unknown sample (in replicates) into an appropriate labeled tube. Also include a “blank”, containing only the buffer used for the preparation of samples and standards.
- Add 2.0ml of the BCA Working Solution (see prior to use) and mix well.
- Cover the tubes and incubate in a water bath at 37ºC for 30 minutes
(or alternatively at room temperature for 2 hours). - Allow to cool to room temperature
- Set the spectrophotometer, according to the manufacturer´s instructions to read at 562nm.
- Zero the instrument using a cuvette filled with ddH2O (this is not the “blank”!)
- Read the absorbance of the blank, all the standards, and samples in the shortest possible time, preferably within 15 minutes. (The color development continues even at room temperature, which may affect accuracy if measurements are delayed).
Protocol for Microplate Reader
- Pipet 25µl of each standard and unknown sample (in replicates) into an appropriate labeled tube. Also include a “blank”, containing only the buffer used for the preparation of samples and standards.
- Add 200µl of the BCA Working Solution (see prior to use) and mix well using a plate shaker for 30 sec.
- Cover the plate and incubate at 37ºC for 30 minutes
- Allow to cool to room temperature
- Read the absorbance at 562nm (or near 562nm) according to the manufacturer´s instructions.
Quantification
- Determine the average value of the “blank” standard replicates and subtract this value from all other measurements.
- Plot the average “blank-corrected” values for each protein standard against its known concentration (in µg/ml), and construct a standard calibration curve.
- Use the standard curve to determine the protein concentration of each unknown sample and calculate the original sample concentration taking into account all additional dilution steps, if carried out.
In case the correction factor for the protein in the unknown sample is known (see page 2), one should divide the final outcome by the correction factor in order to prevent over or underestimation.
