BCA Protein Colorimetric Detection Kit

Catalog No. ABIN2815083

Product Details

Target: Bicinchoninic Acid

Detection Method: Colorimetric

Application: Biochemical Assay (BCA), Quantification (Q)

Purpose: The DetectX® BCA Protein Assay Kit is designed to quantitatively measure total protein content in a variety of samples. The assay measures all types of proteins from all species.

Brand: DetectX®

Sample Type: Serum, Plasma, Tissue Homogenate, Cell Lysate

Analytical Method: Quantitative

Components: Clear 96 well Half Area Plates 2 Plates Corning Costar Plate 3695.
Bovine Serum Albumin Standard 200 μL Bovine Serum Albumin (BSA) at 10 mg/mL BCA Reagent 16 mL BCA Reagent solution
BCA Enhancer 320 μL BCA Enhancer solution containing copper sulfate
Plate Sealers 2 Each

Material not included: Deionized water.
Repeater pipet with disposable tips capable of dispensing 75 μL. 96 well plate reader capable of reading optical absorption at 560 nm.
Software for converting optical density (OD) readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting.

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Protocol: A bovine serum albumin (BSA) standard is provided to generate a standard curve for the assay and all samples should be read off of the standard curve.
Samples are diluted in water and added to the wells.
The BCA Color Solution is made by mixing the BCA Reagent with the BCA Enhancer.
The BCA Color Solution is added to all wells and the plate incubated at 37 °C.
Protein in the samples reacts with the BCA Color Reagent to generate a purple colored product which is read at 560 nm.

Assay Procedure:

1. Pipet 10 µL of samples or appropriate standards into duplicate wells in the plate. 2. Pipet 10 µL of water into duplicate wells as the Zero standard. 3. Add 75 µL of the BCA Color Solution to each well using a repeater pipet. 4. Seal the plate and incubate at 37 °C for 2 hours. 5. Read the optical density at 560 nm.

Calculation of Results:

Average the duplicate OD readings for each standard and sample.
Create a standard curve by reducing the data using computer software capable of generating a four-parameter logistic curve (4PLC) fit.
The sample concentrations obtained should be multiplied by the dilution factor to obtain neat sample values.

Assay Precision: Three samples diluted in water were run in replicates of 20 in an assay.
Inter Assay Precision:
- Regular Format Three samples diluted in water were run in duplicates in thirteen assays run over multiple days by three operators.

Restrictions: For Research Use only

Handling

Precaution of Use: As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
The complete insert should be read and understood before attempting to use the product.
The Bovine Serum Albumin (BSA) standard contains low concentrations (≤0.9%) of sodium azide.
Please dispose of any solutions using this material with copious amounts of water. ® www.ArborAssays.com 5 WEB INSERT 150617 Sample typeS and preparatiOn Samples should be diluted in deionized water.
Two protocols are listed to allow most samples to be read and samples with very low levels of protein to be measured.
Dilutions should be made to ensure that protein levels for samples fall within the standard curve range.
Cell lysates in our Cell Lysis Buffer, X050-100ML, should be diluted 1:10 or greater with deionized water to bring the total protein content in the analyzed sample down to 1 mg/mL or lower.
Human serum and plasma samples will typically have between about 60 and 100 mg of total protein per mL.
They should be diluted 1:100 or greater in deionized water to be measured in this kit.
Most urine samples will have levels between 50 to 200 μg/mL.
Urine samples should be diluted at least 1:2 with deionized water prior to testing.
Sample incOmpatibilitieS The assay will tolerate most common laboratory chemicals.
Known incompatibilities include re- ducing agents such as dithioerythritol (DTE), dithiothreitol (DTT) and ß-mercaptoethanol (BME) at concentrations above 1 mM.
Organic solvent content should be below 1%.
Sample cOmpatibilitieS The assay will work with most buffer and other salts at concentrations below 10 mM.
A simple 1:10 dilution in water should bring most buffer salts down to this level.
It is compatible with both urea and guanidine hydrochloride at 3M or lower.
Most detergents including Triton, Tween, SDS, Nonidet, etc below 1% are also compatible.
It is compatible with ammonium sulfate at 1M and with 10% glycerol.
EDTA and EGTA below 2 mM are also compatible.

Handling Advice: Standard preparatiOn - regular fOrmat Standard Preparation BSA Standards are prepared by labeling six tubes as #1 through #6.
Add 180 μL of water to tube number 1 and pipet 100 μL of water into tubes #2 to #6.
Carefully add 20 μL of the BSA Standard Stock to tube #1 and vortex.
Add 100 μL of tube #1 to tube #2 and vortex completely.
Repeat this for tubes #3 through #6.
The concentration of BSA in tubes 1 through 6 will be 1,000, 500, 250, 125, 62.5, and 31.25 μg/mL.
Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Deionized Water (μL) 180 100 100 100 100 100 Addition Stock Std 1 Std 2 Std 3 Std 4 Std 5 Volume of Addition (μL) 20 100 100 100 100 100 Final Conc (μg/mL) 1,000 500 250 125 62.5 31.25 Standard preparatiOn - high SenSitivity Standard Preparation BSA Standards are prepared by labeling six tubes as #1 through #6.
Add 490 μL of water to tube number 1 and pipet 200 μL of water into tubes #2 to #6.
Carefully add 10 μL of the BSA Standard Stock to tube #1 and vortex.
Add 200 μL of tube #1 to tube #2 and vortex completely.
Repeat this for tubes #3 through #6.
The concentration of BSA in tubes 1 through 6 will be 200, 100, 50, 25, 12.5, and 6.25 μg/mL.
Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Deionized Water (μL) 490 200 200 200 200 200 Addition Stock Std 1 Std 2 Std 3 Std 4 Std 5 Volume of Addition (μL) 10 200 200 200 200 200 Final Conc (μg/mL) 200 100 50 25 12.5 6.25 Use all Standards within 2 hours of preparation.

Storage: 4 °C

Storage Comment: All components of this kit should be stored at room temperature until the expiration date of the kit.

Target Details

Target: Bicinchoninic Acid

Alternative Name: BCA

Background: Protein determination is one of the most common operations performed in biochemical research. The principle of the bicinchoninic acid (BCA) assay is similar to the Lowry assay1, and relies on the formation of a Cu2+-protein complex under alkaline conditions2, followed by reduction of the Cu2+ to Cu1+. The amount of reduction is proportional to protein present. It has been shown that cysteine, cystine, tryptophan, tyrosine, and peptide bonds2 are able to reduce Cu2+ to Cu1+. BCA forms a purple-blue complex with Cu1+ in alkaline environments, thus providing a basis to monitor the reduction of alkaline Cu2+ by proteins. The method combines the well-known reduction of Cu2+ to Cu1+ by protein in an alkaline medium (the biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+1) using a unique reagent containing bicinchoninic acid. The purple-colored reaction product of this assay is formed by the chelation of two molecules of BCA with one cuprous ion. This water-soluble complex exhibits a strong absorbance at 562nm that is nearly linear with in- creasing protein concentrations over a broad working range (6-1,000 μg/mL). The BCA chemistry is not a true end-point method, that is, the final color continues to develop. However, following incubation, the rate of continued color development is sufficiently slow to allow large numbers of samples to be assayed together. References 1. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ., "Protein measurement with the Folin phenol reagent". J. Biol. Chem. 1951, 193 (1): 265-75. 2. Smith, PK, et. al., "Measurement of protein using bicinchoninic acid.", Anal. Biochem., 1985, 150: (1), 76-85. ® www.ArborAssays.com 3 WEB INSERT 150617