Bicinchoninic acid Protein Assay Kit for Protein Quantitation and Determination Product code A7787 The Bicinchoninic acid Protein Assay Kit for protein quantitation and determination is formulated for the colorimetric detection and quantitation of total protein in the 20 2,000 µg/ml concentration range. Like the Lowry method, the assay relies on the reduction of Cu 2+ ions by protein. The Cu + thus formed is detected by conversion into a violet-colored substance by reaction with bicinchoninate. The absorbance at 562 nm of the Cu(I)-(bicinchoninate) 2 complex is directly proportional to protein concentration. The bicinchoninic acid protein assay is more sensitive than the Lowry method and less subject to interference. In particular, it is insensitive to detergents such as Triton X-100 and SDS (1 %). Important otes Precipitates which may form in Reagent A or B during storage should be dissolved by gentle warming and stirring. Color development continues even at room temperature. However no significant error is introduced if all absorbance measurements are completed within 10 minutes. Reagents that chelate metal ions, change the ph of the assay or reduce copper, are known to interfere with the assay. Please check that the following components are not in the sample buffer: ascorbic acid, catecholamines, creatinine, cysteine, EGTA, impure glycerol, hydrogen peroxide, hydrazides, iron, lipids, melibiose, phenol red, impure sucrose, tryptophan, tyrosine, uric acid. ther substances affect the assay to a lesser extent and, if their concentration in the sample buffer is below a certain value, can be tolerated. Please refer to Table 2 for maximum compatible concentrations of many of these substances. It is necessary to create a standard curve during each assay regardless of the format used. The Bicinchoninic acid Protein Assay working solution is stable for several days at RT. If not used immediately, it should be stored at RT in a closed container. Required Equipment/Materials Spectrophotometer capable of measuring absorbance in the region of 560 nm Water bath Test tubes or 96 well plate Protein Standards rdering information Prod. o. Size (sufficient for) Reagent A (Bicinchoninic acid /tartrate in alkaline carbonate buffer) Bicinchoninic acid Protein Assay Reagent B (4 % CuS 4 5H 2 in H 2 ) A7787,SAMPLE may vary A7787,SAMPLE 50 ml A7787,SAMPLE 5 ml X A7787,0500 500 tests A7787,0500A 4 x 25 ml A7787,0500B 2 x 15 ml X Protocol Storage: Shipment: Stability (Reagents A + B): Stability (working solution): Sensitivity: o. of tests RT ambient temperature 2 years several days 20-2,000 µg/ml protein 500 test tubes or 5,000 microplate assays 1
Procedures Preparation of the Working Reagent Mix 50 Parts of Reagent A and 1 part of Reagent B. The amount of Working Reagent required for each sample is 2.0 ml f or the Test Tube Procedure and 200 µl for the Micro assay Plate Procedure. A precipitate is initially formed when reagent C (CuS 4 ) is added to the mixture of Reagents A and B. It will then redissolve to yield a clear, apple green Working Reagent. Prepare sufficient volume of Working Reagent for the samples to be assayed plus the calibration standards. For example, for the standard test tube procedure with 9 standards (including a blank), 3 unknowns and 2 replicates for each sample, 48 ml of Working reagent is required. The Working Solution is stable for several days when stored at room temperature in a closed container. Preparation of the Calibration Standards Prepare a fresh set of protein standards in the 20-2,000 µg/ml range, preferably using the same diluent as your sample. While the most accurate results are obtained using a pure sample of the protein to be measured as standard, in many cases this is expensive or not available. Therefore, the standards are generally prepared from a 1.0-2.0 mg/ml stock solution of Bovine Serum Albumin. For several proteins a Correction Factor relative to BSA is reported in the following table: Protein Correction Factor Protein Correction Factor Albumin, bovine serum 1.00 IgG, mouse 1.18 Aldolase, rabbit muscle 0.85 IgG, rabbit 1.12 α-chymotrypsinogen, bovine 1.14 IgG, sheep 1.17 Cytochrome c, horse heart 0.83 Insulin, bovine pancreas 1.08 γ-globulin, bovine 1.11 Myoglobin, horse heart 0.74 IgG, bovine 1.21 valbumin 0.93 IgG, human 1.09 Transferrin, human 0.89 A. Test tube Procedure.B. The test tube procedure requires a larger volume (0.1 ml) of protein sample but, since the sample to working reagent ratio is 1/20, the effect of interfering substances is minimized. 1.) Pipette 0.1 ml of each standard (including a blank) and unknown sample into a labelled test tube. 2.) Add 2.0 ml of Working Reagent to each tube and mix thoroughly. 3.) Close / Cover the tubes and incubate at a selected temperature and time in a water bath: working range: Standard Protocol: RT Protocol: working range: Enhanced Protocol: 4.) Cool the tubes to RT. 20 2,000 µg/ml 37 C for 30 min RT for 2 h 5 250 µg/ml 60 C for 30 min 5.) Measure the absorbance at 562 of all the samples within 10 minutes. 6.) Subtract the 562 nm absorbance value of the Blank from the readings of the standards and the unknowns. 7.) Plot the blank-corrected 562 nm reading for each standard vs. its concentration. Determine the protein concentration of each unknown from the calibration plot. 2
B. Microplate Procedure.B. The Microplate Procedure requires a smaller volume (10-25 μl) of protein sample but, since the sample to working reagent ratio is 1/8, it offers less flexibility in overcoming the effect of interfering substances and obtaining a low levek of detection. 1.) Pipette a 25 µl of each standard (including a blank) and unknown sample into a microwell plate. 2.) Add 200 µl of Working Reagent to each well and mix thoroughly. 3.) Cover the plate and incubate at 37 C for 30 min. Do not incubate at higher temperatures. 4.) Cool plate to RT. 5.) Measure the absorbance at 562 of all the samples on a plate reader. 6.) Subtract the 562 nm absorbance value of the blank from the readings of the standards and the unknowns. 7.) Plot the Blank-corrected 562 nm reading for each standard vs. its concentration. Determine the protein concentration of each unknown from the calibration plot. 3
Compatibility of Reagents with the Standard Test Tube Protocol Salts & Buffers Concentration Reducing & Thiol Containing Agents Concentration ACES, ph 7.8 10 mm -Acetylglucosamine in PBS C Ammonium sulfate C Ascorbic acid C Bicine, ph 8.4 2 mm Cysteine C Bis-Tris, ph 6.5 0.2 mm Dithioerythritol (DTE) C Calcium chloride in TBS, ph 7.2 10 mm Dithiothreitol (DTT) C a-carbonate/a-bicarbonate (0.2 M), ph 9.4 undiluted Glucose 1 mm Cesium bicarbonate 100 mm 2-Mercaptoethanol 1 mm CHES, ph 9.0 100 mm Thimerosal C a-citrate (0.6 M),a-Carbonate (0.1 M), ph 9.0 1:600 dilution* Misc. Reagents & Solvents a-citrate (0.6 M), MPS (0.1 M), ph 7.5 1:600 dilution* Acetone 1.0 % Cobalt chloride in TBS, ph 7.2 C Acetonitrile 1.0 % EPPS, ph 8.0 100 mm Aprotinin 1 mg/l Ferric chloride in TBS, ph 7.2 0.5 mm DMF, DMS 1.0 % Glycine HCl (ph 2.8) 100 mm Ethanol 1.0 % Guanidine HCl 4 M Glycerol (fresh) 1.0 % HEPES, ph 7.5 100 mm Hydrazide C Imidazole, ph 7.0 12.5 mm Hydrides (a 2BH 4 or acbh 3) C MES, ph 6.1 100 mm Hydrochloric acid 10 mm MES (0.1 M), acl (0.9 %), ph 4.7 1:4 dilution* Leupeptin 10 mg/l MPS, ph 7.2 100 mm Methanol 1.0 % Modified Dulbecco s PBS, ph 7.4 undiluted Phenol Red C ickel chloride in TBS, ph 7.2 0.2 mm PMSF 1 mm PBS; Phosphate (0.1 M), acl (0.15 M) ph 7.2 undiluted Sodium hydroxide 50 mm PIPES, ph 6.8 100 mm Sucrose 4 % RIPA lysis buffer; 50 mm Tris, 150 mm 1:10 dilution* TLCK 0.1 mg/l acl, 0.5 % DC, 1 % P-40, 0.1 % TPCK 0.1 mg/l SDS, ph 8.0 Urea 3 M Sodium acetate, ph 4.8 200 mm o-vanadate (sodium salt), in PBS, ph 7.2 1 mm Sodium azide 0.2 % Detergents Sodium bicarbonate 100 mm Brij 35 5.0 % Sodium chloride 1 M Brij 56, Brij 58 1.0 % Sodium citrate, ph 4.8 (or ph 6.4) 5 mm (16.7 mm) CHAPS (CHAPS) 1.0 % (5.0 %) Sodium phosphate 100 mm Deoxycholic acid 5.0 % Tricine, ph 8.0 2.5 mm onidet P-40 (P-40) 5.0 % Triethanolamine, ph 7.8 0.5 mm ctyl -D-glucoside 0.1 % Tris 50 mm ctyl -D-thioglucopyranoside 5.0 % TBS; Tris (25 mm), acl (0.15 M), ph 7.6 1:10 dilution* SDS 5.0 % Tris (25 mm), Glycine (192 mm), ph 8.0 1:10 dilution* Span 2 1.0 % Tris (25 mm), Glycine (192 mm), SDS (0.1 %), ph 8.3 undiluted Triton X-100 5.0 % Zinc chloride in TBS, ph 7.2 0.5 mm Triton X-114 0.05 % Chelating agents Triton X-305, Triton X-405 1.0 % EDTA 0.5 mm Tween 20, Tween 80 5.0 % EGTA C Tween 60 0.5 % Sodium citrate, ph 4.8 (or ph 6.4) 5 mm (16.7 mm) *Diluted with ultrapure water C: not compatible Zwittergent 3-14 C 4
Methods for eliminating/reducing the effect of interfering substances Remove the interfering substance by dialysis or gel filtration. Dilute sample. This works only if the starting protein is sufficiently concentrated to remain in the working range upon dilution. Precipitate the proteins in the sample with acetone or trichloroacetic acid. The protein pellet is then solubilized in ultrapure water or directly in the Working Reagent. Increase the amount of copper in the Working Reagent (e.g. use a greater proportion of Reagent B; e.g. Reagent A:B in a ratio of 50:2 or 50:3). ote: For greatest accuracy, the protein standards must be treated identically to the sample(s). Troubleshooting bservation Possible Cause Precautions/Remedy o color development Chelating agents are present in the sample buffer Sample color less intense than expected Sample color is darker than expected All the tubes are dark purple Persistent precipitation appears after Reagent C (CuS 4 ) is added to Reagents A+B ph is altered by strong acid or alkaline buffer Protein concentration is too high Lipids or lipoproteins are present in the sample buffer Reducing agents are present in the sample buffer Thiols are present in the sample buffer Temperature of reagents is too low. Dialyze or desalt the sample. Dilute the sample. Dialyze or desalt the sample. Dilute the sample. Dilute the sample Add 2 % SDS to the sample to eliminate interference from lipids Dialyze or dilute the sample Dialyze or dilute the sample Gently warm (~37 C) all reagents immediately prior to mixing. r slightly dilute the Working Reagent with deionized water until precipitate disappears. Also, the WR solution could simply be filtered (the loss of reagents would be negligible). 5
The Bicinchoninic acid Protein Quantitation and Determination Products Protein Quantitation and Determination Bicinchoninic acid Protein Assay and Bicinchoninic acid Protein Assay Micro kits are formulations based on bicinchoninic acid for the rapid and sensitive detection and quantitation of total protein content. The Bicinchoninic acid method is faster and easier than Lowry, with much greater tolerance to interference from non-ionic detergents and buffer salts. The Bicinchoninic acid method combines the biuret reaction, i.e., the reduction of Cu 2+ ions to Cu + by proteins in an alkaline medium with complexation of the latter with bicinchoninic acid. The purple-colored Cu(bicinchoninic acid) 3-2 complex displays a strong absorbance at 562 nm which is proportional to protein concentration over a broad working range (20-2,000 μg/ml for Bicinchoninic acid Protein Assay and 0.5-20 μg/ml for Bicinchoninic acid Protein Assay Micro). - - Protein + Cu 2+ H - (biuret reaction) Cu + + bicinchonic acid Cu + - - Purple Complex of Cu + with BCA Protein concentrations are generally determined with reference to standards of a common protein such as Bovine Serum Albumin (BSA). If a more accurate quantitation of an unknown protein is required, the calibration curve is constructed using a protein similar to the unknown. Special features of the Bicinchoninic acid Protein Assay and Bicinchoninic acid Protein Assay Micro Photometric method (reading at 562 nm) More sensitive than Lowry and less subject to interferences. In particular, it is insensitive to detergents such as Triton -X 100 and SDS (1 %) Linear working range for BSA is 20-2,000 μg/ml for the Bicinchoninic acid Protein Assay and 0.5-20 μg/ml for the Bicinchoninic acid Protein Assay Micro Less protein to protein variation than Coomassie -based assays Reagents are stable two years at room temperature Adaptable to microplate format References 1.) Smith, P.K. et al. (1985) Anal. Biochem. 150, 76-85 Measurement of protein using bicinchonininc acid. 2.) Walker, J. M. (1994) Methods. Mol. Biol. 32, 5-8. 3.) Pingoud, A, Urbanke, C., Hoggett J., Jeltsch, A., Biochemical Methods, pp.157-159, Wiley-VCH 2002. Version WM100311 6