Coomassie Plus Protein Assay Reagent Kit

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1 INSTRUCTIONS Coomassie Plus Protein Assay Reagent Kit N. Meridian Road P.O. Box 117 Rockford, IL w Product Description The Coomassie Plus Protein Assay Reagent Kit contains sufficient reagents to perform 630 test tube assays or 3,160 microwell plate assays. The kit contains: Coomassie Plus Protein Assay Reagent, 950 ml, containing the Coomassie dye, methanol, phosphoric acid and solubilizing agents in water. Caution: Phosphoric acid is a corrosive liquid. Albumin Standard, 10 x 1 ml ampules, containing bovine serum albumin (BSA) at a concentration of 2.0 mg/ml in a solution of 0.9% saline and 0.05% sodium azide. Available separately as product # If any kit reagent shows discoloration or evidence of microbial contamination, discard the reagent. Note: If you are working with IgG samples, you may prefer our Bovine Gama Globulin (BGG) Standard (Prod. No , 10 x 1 ml ampules, 2 mg/ml). This BGG standard may be diluted and used as described for the BSA standard included in this kit (Table 1). If you would like to avoid the tedious and time consuming step of preparing the diluted protein standards, Pierce also offers both the BSA and BGG standards in sets containing 7 x 3.5 ml bottles of pre-diluted protein standards covering the range from 125 µg/ml to 2,000 µg/ml. The Pre-Diluted Protein Standards are available separately as Prod. No (BSA) or (BGG). Introduction: Coomassie Plus Protein Assay Reagent The Pierce Coomassie Plus Protein Assay Reagent is a quick and ready-to-use modification of the well-known Bradford, Coomassie, dye-binding, colorimetric method for total protein quantitation. 1 This Bradford reagent modification greatly reduces the tendency of Coomassie dye containing reagents to give a characteristically non-linear response curve. The Coomassie Plus Reagent results in a substantially more linear response curve in a defined range of protein concentration. In addition, our special formulation results in significantly less protein-to-protein variation than is observed with other Bradford type Coomassie dye based formulations. When Coomassie binds protein in an acidic medium, an immediate absorbance shift occurs from 465 nm to 595 nm with a simultaneous color change of the reagent from green/brown (or red/brown) to blue. The Coomassie - Protein Reaction Scheme Protein + Coomassie G-250 in an acidic medium oprotein-dye complex (blue color, measured at 595 nm) Preparation 1. Preparation of Diluted BSA Standards Prepare a fresh set of protein standards by diluting the 2.0 mg/ml BSA stock standard (Stock) (preferably in the same diluent as your samples) as illustrated in Table 1. A 1 ml ampule of the 2.0 mg/ml BSA standard is sufficient to prepare a set of diluted standards for either working range. There will be sufficient volume for three replications of each diluted BSA standard, if that is desired. 2. Mixing of the Coomassie Plus Protein Assay Reagent Allow the Coomassie Plus Reagent to come to room temperature. Mix the Coomassie Plus Reagent solution just prior to use by gently inverting the bottle several times. Do not shake. 1

2 The Standard Protocol: Test Tube Version (Working Range = 100 to 1,500 µg/ml; linear working range with BSA = 125-1,000 µg/ml; linear working range with IgG = 125-1,500 µg/ml) 1. Pipet 0.05 ml of each standard or unknown sample into appropriately labeled test tubes. Use 0.05 ml of the diluent for the blank tubes. 2. Add 1.5 ml of the Coomassie Plus Reagent to each tube, mix well. 3. Measure the absorbance at 595 nm of each tube versus a water reference. concentration in µg/ml. Using the standard curve, determine the protein concentration for each unknown sample. The Standard Protocol: Microwell Plate Version (Working Range = 100 to 1,500 µg/ml) 1. Pipet 10 µl of each standard or unknown sample into the appropriate microwell plate wells. Use 10 µl of the diluent for the blank wells. 2. Add 300 µl of the Coomassie Plus Reagent to each well, mix the plate on a plate shaker for 30 seconds. 3. Measure the absorbance at or near 595 nm on a plate reader. concentration in µg/ml. Using the standard curve, determine the protein concentration for each unknown sample. Note: When compared to the standard tube protocol, the 595 nm readings obtained with the microwell plate protocol are lower due to the shorter light path employed with the plate reader. This may increase the minimum detection level of the assay. If higher 595 nm readings are required, use 15 µl of standard or sample and 300 µl of working reagent per well. The Micro Protocol: Test Tube Version (Working Range = 1 to 25 µg/ml) 1. Pipet 1.0 ml of each standard or unknown sample into appropriately labeled test tubes. Use 1.0 ml of the diluent for the blank tubes. 2. Add 1.0 ml of the Coomassie Plus Reagent to each tube, mix well. 3. Measure the absorbance at 595 nm of each tube versus a water reference. concentration in µg/ml. Using the standard curve, determine the protein concentration estimate for each unknown sample. The Micro Protocol: Microwell Plate Version (Working Range = 1 to 25 µg/ml) 1. Pipet 150 µl of each standard or unknown sample into the appropriate microwell plate wells. Use 150 µl of the diluent for the blank wells. 2. Add 150 µl of the Coomassie Plus Reagent to each well, mix the plate on a plate shaker for 30 seconds. 3. Measure the absorbance at or near 595 nm on a plate reader. concentration in µg/ml. Using the standard curve, determine the protein concentration estimate for each unknown sample. 2

3 Performance characteristics of the Coomassie Plus Protein Assay Reagent 1. Color response curves with BSA and bovine gamma globulin (BGG) Typical response curves for BSA and Bovine gamma globulin (BGG) BSA BGG Protein concentration in µg/ml 2000 Figure 1 shows the typical color response curve for Bovine serum albumin (BSA) and Bovine Gamma Globulin (BGG) using the Standard Test Tube Protocol. 2. Protein-to-protein variation All proteins are unique and their varying structures can give variable responses. Each of the commonly used assay methods exhibit some degree of varying color response toward different proteins. These differences relate to dissimilarity among proteins due to amino acid sequence, pi, structure and the presence of certain side chains or prosthetic groups that can dramatically alter the protein s color response. Most protein assay methods utilize bovine serum albumin (BSA) or immunoglobulin (IgG) as the standard against which the concentration of protein in the sample is determined. Using either of these proteins as the standard works well in most assay methods. However, if great accuracy is required, the standard curve must be prepared from a pure sample of the target protein to be measured. If a pure sample of the target protein is not available, select the standard protein from those proteins that generate a color response that is close to the color response of the target protein. Table 2 shows the protein-to-protein variation in color response of the Coomassie Plus Protein Assay Reagent for thirteen proteins compared to the color response for BSA. All proteins were tested at a concentration of 1,000 µg/ml using the Standard Test Tube Protocol. The average net color response for BSA was normalized to 1.00 and the average net color response of the other proteins is expressed as a ratio to the response of BSA. The protein-to-protein variation observed with the Coomassie Plus Reagent is significantly less than that seen with other Bradford-type Coomassie formulations. 3. Compatible substances The substances listed in Table 3 were found to be compatible with the Coomassie Plus Protein Assay Reagent if the error in the estimate of the protein concentration (BSA at 1,000 µg/ml) caused by the presence of the substance in the sample was less than or equal to 10%. The blank corrected 595 nm readings (for the 1,000 µg/ml BSA standard + substance) were compared to the net 595 nm of the 1,000 µg/ml BSA standard prepared in 0.9% saline. 4. Substances known to interfere Both ionic and nonionic detergents at high concentrations are known to interfere and cause precipitation with the Coomassie Plus Protein Assay Reagent. Troubleshooting a. The presence of incompatible substances in the sample Interference in the Coomassie Plus Protein Assay may be eliminated or overcome by: 1). removing the interfering substance by dialysis, gel filtration, or protein precipitation 3

4 2). diluting the sample to the point that the substance no longer interferes (This works if the starting protein concentration of the sample is high.) b. Reading at wavelengths other than 595 nm If a photometer or plate reader is not available with a 595 nm filter, the blue color may be measured at any wavelength between 570 nm and 610 nm. The maximum sensitivity of the assay occurs when the absorbance of the Coomassie -protein complex is measured at 595 nm. Taking the absorbance measurements at any wavelength other than 595 nm will result in a lower slope for the standard curve and may increase the minimum detection level for the protocol. c. Cleaning and reuse of glassware Care must be exercised when cleaning glassware that will be used again for protein assays. Thorough cleaning often requires the use of a detergent which must be completely removed in the final rinse. Also, the Coomassie dye will stain glass or quartz cuvettes. The cuvettes can be easily cleaned with our detergent concentrate cleaning agent, PC-54 (#72228), followed by a thorough final rinsing with deionized water. If you prefer, disposable polystyrene cuvettes may be used to eliminate the cuvette cleaning chore. d. Effect of temperature on the 595 nm readings The absorbance readings at 595 nm obtained with the Coomassie Plus Reagent are dependent on the temperature of the reagent to some extent. As the reagent temperature increases to room temperature, the 595 nm readings will increase. Therefore, it is important that the Coomassie Plus Reagent be at room temperature during the assay. e. Dye-dye and dye-protein aggregate formation in the Coomassie Plus Reagent The Coomassie Plus Protein Assay Reagent contains additives that help to slow down the formation of dye-dye and dyeprotein aggregates that occurs in all Coomassie dye based protein reagents. Over time, if left undisturbed, the aggregates with become large enough to be easily visible. When left overnight in a clear glass tube, the reagent forms a dye-dye aggregate that is visible as a dark precipitate of dye in the bottom of the tube with nearly colorless liquid over the dye aggregate. The dye-dye aggregates can form within hours in the stored reagent while the dye-protein-dye aggregates seem to form even more quickly. Fortunately, gentle mixing completely disperses the aggregates. Therefore, it is good practice, with all Coomassie based protein assay reagents, to mix the Coomassie Plus Reagent prior to pipetting and to again mix each tube or plate just before the 595 nm readings are taken. References 1. Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, Davies, E.M.(1988). Protein assays: A review of common techniques, Amer. Biotech. Lab Compton, S.J. and Jones, C.J. (1985) Mechanism of dye response and interference in the Bradford protein assay. Anal. Biochem. 151, Tal, M., S ilberstein, A. and Nusser, E. (1980). Why does Coomassie brilliant blue R interact differently with different proteins? J. Biol. Chem. 260, Sedmak, J.J. and Grossberg, S.E. (1977). A rapid, sensitive and versatile assay for protein using Coomassie brilliant blue G-250. Anal. Biochem. 79, Sorensen, K. (1994). Coomassie protein assay used for quantitative determination of sodium cyanoborohydride (NaCNBH 3 ). Anal. Biochem. 218, Sherwood, J.K., Zeitlin, L., Whaley, K.J., Cone, R.A. and Saltzman, M. (1996). Controlled release of antibodies for long-term topical passive immunoprecipetation of female mice against genital herpes. Nature Biotech. 14, Coomassie, Tween, and Brij are registered trademarks of ICI Americas. Triton and Lubrol are registered trademarks of Rohm & Haas. Zwittergent is a registered trademark of Calbiochem-Novabiochem Corp. Pierce Chemical Company, 9/1999. Printed in the U.S.A. 4

5 Table 1: Preparation of the Diluted BSA Standards Standard Test Tube or Plate Protocol Working Range = µg/ml Volume of the BSA to Add 300 µl of (Stock) 375 µl of (Stock) of (Stock) 175 µl of (A) of (B) of (D) of (E) 100 µl of (F) Volume of Diluent to Add 0 µl 125 µl 175 µl 400 µl Final BSA Concentration 2,000 µg/ml 1,500 µg/ml (A) 1,000 µg/ml (B) 750 µg/ml (C) 500 µg/ml (D) 250 µg/ml (E) 125 µg/ml (F) 25 µg/ml (G) Micro Test Tube or Plate Protocol Working Range = 1-25 µg/ml Volume of the BSA to Add 30 µl (Stock) 50 µl (Stock) 30 µl (Stock) 2,500 µl (a) 2,000 µl (b) 1,500 µl (c) Volume of Diluent to Add 2,370 µl 4,950 µl 3,970 µl 2,500 µl 2,000 µl 1,500 µl Final BSA Concentration 25 µg/ml 20 µg/ml (a) 15 µg/ml 10 µg/ml (b) 5 µg/ml (c) 2.5 µg/ml Table 2: Protein-to-Protein Variation Coefficient of variation = (standard deviation / average ratio) X 100 Protein Tested avg. test net Abs. Ratio = avg. BSA net Abs. Albumin, bovine serum Aldolase, rabbit muscle Chymotrypsinogen, bovine Cytochrome C, horse heart Gamma globulin, bovine IgG, bovine IgG, human IgG, mouse IgG, rabbit IgG, sheep Insulin, bovine pancreas Myoglobin, horse heart Ovalbumin Transferrin, human Average ratio = Standard deviation = Coefficient of variation = Prod. #23236 Coomassie Plus Reagent 595 nm % 5

6 Table 3: Substances found to be compatible with the Coomassie Plus Protein Assay Reagent using the Standard Test Tube Protocol: (Amount listed refers to the actual concentration in the sample.) Substance Compatible Concentration Detergents Brij % Brij % Brij % CHAPS 5.0% CHAPSO 5.0% Deoxycholic acid 0.04% Lubrol PX 0.031% Octyl b-glucoside 0.50% Nonidet P-40 (NP-40) 0.50% Octyl b-thioglucopyranoside 3.0% SDS 0.016% SPAN % Triton X % Triton X % Triton X % Triton X % Tween % Tween % Tween % Zwittergent % Chelating agents EDTA EGTA 2 mm Sodium citrate 200 mm Reducing & Thiol Containing Agents N-acetylglucosamine in PBS, ph 7.2 Ascorbic acid 50 mm Cysteine Dithioerythritol (DTE) 1 mm Dithiothreitol (DTT) 5 mm Glucose 1.0 M Melbiose 2-Mercaptoethanol 1.0 M Potassium thiocyanate 3.0 M Thimerosal 0.01% Salts/Buffers ACES, ph 7.8 Ammonium sulfate 1.0 M Asparagine Bicine, ph 8.4 Bis-Tris, ph 6.5 Borate (50 mm), ph 8.5 (Prod. #28384) undiluted B-PER Reagent 1:2 dilution* Calcium chloride in TBS, ph 7.2 Na-Carbonate/Na-Bicarbonate (0.2 M), ph 9.4 (Prod. #28382) undiluted Cesium bicarbonate CHES, ph 9.0 Na-Citrate (0.6 M), Na-Carbonate (), ph 9.0 (Prod. #28388) undiluted Substance Compatible Concentration Salts/Buffers continued Cobalt chloride in TBS, ph 7.2 EPPS, ph 8.0 Ferric chloride in TBS, ph 7.2 Glycine Guanidine HCl 3.5 M HEPES, ph 7.5 Imidazole, ph mm MES, ph 6.1 MES (), NaCl (0.9%), ph 4.7 undiluted MOPS, ph 7.2 Modified Dulbecco s PBS, ph 7.4 undiluted Nickel chloride in TBS, ph 7.2 PBS; Phosphate (), NaCl (0.15 M), undiluted ph 7.2 (Prod. #28372) PIPES, ph 6.8 RIPA lysis buffer; 50 mm Tris, 150 mm NaCl, 1:40 dilution* 0.5% DOC, 1% NP-40, 0.1% SDS, ph 8.0 Sodium acetate, ph mm Sodium azide 0.50% Sodium bicarbonate Sodium chloride 5.0 M Sodium citrate, ph 4.8 or ph mm Sodium phosphate Tricine, ph 8.0 Triethanolamine, ph 7.8 Tris 2.0 M TBS; Tris (25 mm), NaCl (0.15 M), ph undiluted 7.6 (Prod. #28376) Tris (25 mm), Glycine (192 mm), undiluted ph 8.0 Prod. #28380) Tris (25 mm), Glycine (192 mm), SDS 1:4 dilution* (0.1%), ph 8.3 (Prod. #28378) Zinc chloride in TBS, ph 7.2 Misc. Reagents & Solvents Acetone 10% Acetonitrile 10% Aprotinin 10 mg/l DMF 10% DMSO 10% Ethanol 10% Glycerol (Fresh) 10% Hydrochloric Acid Leupeptin 10 mg/l Methanol 10% Phenol Red 0.5 mg/ml PMSF 1 mm Sodium Hydroxide Sucrose 10% TLCK 0.1 mg/l TPCK 0.1 mg/l Urea 3.0 M o-vanadate (sodium salt), in PBS, ph mm 6

A rapid, sensitive protein assay for the accurate analysis of protein concentrations

WHITE PAPER A rapid, sensitive protein assay for the accurate analysis of protein concentrations Protein quantitation is an integral part of many laboratory workflows and often a necessary step before