Dual-Range TM BCA Protein Assay Kit

Transcription

1 Manual DualRange TM BCA Protein Assay Kit BC31/BC325/BC35 V3.1 For Research Use Only Introduction DualRange TM BCA Protein Assay Kit is a perfect protein assay method. It based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein in the 5 2, 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 violetcolored substance by reaction with bicinchoninate. DualRange TM BCA Protein Assay Kit is compatible with many detergents but not compatible with reducing agents, such as DTT, DTE, and 2Mercaptoethanol etc. Product Components DualRange TM BCA Protein Assay Kit Reagent A Reagent B Albumin Standard (2 mg/ml) User s manual BC31 BC325 BC35 1 ml 2 ml 1mL 25 ml 12 ml 1mL x 4 5 ml 12 ml 1mL x1 Safety Information Please wear gloves, lab coat and goggles while operating. Prevent contact product directly. In case of contacting, wash with large amount of water. Storage DualRange TM BCA Protein Assay Kit could be shipped at room temperature. Reagent A and Reagent B should be stored at room temperature for 24 months, and Albumin Standard should be stored at 4 o C. Expiration date is labeled on the bottle or box. Page 1/6

2 Materials needed but not provided 1. Spectrophotometer capable of measuring absorbance in the region of 562 nm (If a 562 nm filter is not available, perform measurement with a 5457 nm filter. Doing so will have no effect on quantification) 2. Water bath 3. Plate Reader 4. Test tubes well plate Instruction A. Preparation of the Working Reagent 1. Prepare Working Reagent by mixing 5 parts of Reagent A and 1 part of Reagent B. 2. The required Working Reagent for each samples of Test Procedure is 2. ml and that of the Microplate Procedure is 2 μl. NOTE: The Working Reagent is a clear, apple green solution and the Working Reagent is stable for several days when stored in a closed container at room temperature. B. Preparation of the Protein Standards 1. Preparation of diluted protein standards: prepare a set protein standards. NOTE: For Test Procedure, use standard guide of 22 μg/ml in Table 1 for the standard protocol and 525 μg/ml in Table 2 for the enhanced protocol. For Microplate Procedure, use standard guide of 22 μg/ml in Table 3 for the standard protocol and 525 μg/ml in Table 4 for the enhanced protocol. Table 1. Preparation of Diluted Albumin (BSA) Standards for Test tube Procedure (working range: 22 μg/ml) Volume of Volume and source of Final BSA Standard Diluent (μl) protein Standards (μl) Concentration (μg/ml) A 3 of Stock 2 B of Stock 15 C of Stock 1 D of tube B dilu on 75 E of tube C dilu on 5 F of tube E dilu on 25 G of tube F dilu on 125 H 4 1 of tube G dilu on 25 I 4 Page 2/6

3 Table 2. Preparation of Diluted Albumin (BSA) Standards for Test tube Proce Volume of Diluent (μl) Volume and source of protein Standards (μl) Final BSA Standard Concentration (μg/ml) dure (working range: 525 μg/ml) A 7 1 of Stock 25 B 4 4 of tube A dilution 125 C 45 3 of tube B dilution 5 D 4 4 of tube C dilution 25 E 4 1 of tube D dilution 5 F 4 Table 3. Preparation of Diluted Albumin (BSA) Standards for Microplate Proce Volume of Diluent (μl) Volume and source of protein Standards (μl) Final BSA Standard Concentration (μg/ml) dure (working range: 22 μg/ml) A 6 of Stock 2 B 4 8 of Stock 15 C 6 6 of Stock 1 D 6 6 of tube B dilution 75 E 6 6 of tube C dilution 5 F 6 6 of tube E dilution 25 G 6 6 of tube F dilution 125 H 24 6 of tube G dilution 25 I 6 Table 4. Preparation of Diluted Albumin (BSA) Standards for Microplate Proce Volume of Diluent (μl) Volume and source of protein Standards (μl) Final BSA Standard Concentration (μg/ml) dure (working range: 525 μg/ml) A 7 1 of Stock 25 B 4 4 of tube A dilution 125 C 45 3 of tube B dilution 5 D 4 4 of tube C dilution 25 E 4 1 of tube D dilution 5 F 4 C. Test tube Procedure 1. Prepare Working Reagent by mixing 5 parts of Reagent A and 1 part of Reagent B. NOTE: Certain substances are known to interfere with the BCA assay and it must be avoided in the sample s buffer. The maximum compatible concentrations for these substances are listed in Table 5. Page 3/6

4 C. Test tube Procedure (~continued) 2. Add 2. ml of the Working Reagent to each tube and mix well. 3. Cover the tubes and incubate at selected temperature and time in a water bath. Working range: 2 2, μg/ml Standard Protocol: 37 C for 3 minutes RT Protocol: RT for 2 hours Working range: 5 25 μg/ml Enhanced Protocol: 6 C for 3 minutes 4. Cool all tubes to room temperature. 5. Turn on the spectrophotometer and set to 562nm, to measure the absorbance of all the samples and the BSA standard. NOTE: All the samples and BSA standard must be measured within 1 minutes to avoid significant error of measurements. NOTE: If a 562 nm filter is not available, perform measurement with a 5457 nm filter. Doing so will have no effect on quantification. 6. Prepare a standard curve by 562 nm BSA measurement and determine the protein concentration of each unknown sample by standard curve. D. Microplate Procedure 1. Pipette 25μL of each standard (Table 3. or Table 4.) or unknown sample replicate into a microplate well. NOTE: If sample size is limited, 1μL of each unknown sample and standard can be used. However, the working range of the assay in this case will be limited to 1252 μg/ml. NOTE: Certain substances are known to interfere with the BCA assay and it must be avoided in the sample s buffer. The maximum compatible concentrations for these substances are listed in Table Add 2μL of the Working Reagent to each well and mix plate thoroughly on a plate shaker for 3 seconds. 3. Cover plate and incubate at a selected temperature and time in a water bath or a thermoshaker. Page 4/6

5 D. Microplate Procedure (~continued) 3. Cover plate and incubate at a selected temperature and time in a water bath or a thermoshaker. Working range: 2 2, μg/ml Standard Protocol: 37 C for 3 minutes RT Protocol: RT for 2 hours Working range: 5 25 μg/ml Enhanced Protocol: 6 C for 3 minutes 4. Cool plate to room temperature. 5. Measure the absorbance at or near 562nm on a plate reader. NOEt: If a 562 nm filter is not available, perform measurement with a 5457 nm filter. Doing so will have no effect on quantification. 6. Prepare a standard curve by measurement the absorbance of BSA at 562nm and determine the protein concentration of each unknown sample by standard curve. Trouble shooting Problem No color development Sample color less intense than expected Sample color is darker than expected All the tubes are dark purple Possible cause Chelating agents are present in the sample buffer ph is altered by strong acid or alkaline buffer Protein concentration is too high Sample contains lipids or lipoproteins Reducing agents are present in the sample buffer Thiols are present in the sample buffer Remedy 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 desalt the sample Dialyze or desalt the sample Appendix Table 5. Compatible concentration of common substances Salts/Buffers Salts/Buffers ACES, ph mM CHES, ph 9. Ammonium sulfate 1.5M Cobalt chloride in TBS, ph 7.2.8mM Asparagine 1mM EPPS, ph 8. Bicine, ph 8.4 2mM Ferric chloride in TBS, ph 7.2 1mM BisTris, ph mM Glycine HCl, ph 2.8 Borate 5mM Guanidine HCl 4M Calcium chloride in TBS, ph 7.2 1mM HEPES, ph 7.5 NaCarbonate/Na Bicarbonate, ph 9.4.2M Imidazole, ph 7. 5mM Cesium bicarbonate MES, ph 6.1 Page 5/6

6 Appendix Table 5. Compatible concentration of common substances (~continued) Salts/Buffers Detergents MOPS, ph 7.2 Modified Dulbecco s PBS, ph 7.4 Nickel chloride in TBS, ph 7.2 PBS; Phosphate (.1 M), NaCl (.15 M), ph 7.2 PIPES, ph 6.8 undiluted 1mM undiluted RIPA lysis buffer; 5mM Tris, 15mM NaCl,.5% DOC, 1% NP 4,.1% SDS, ph 8. undiluted Sodium acetate, ph 4.8 2mM Sodium azide.2% Sodium bicarbonate Sodium chloride Sodium citrate, ph 4.8 or ph 6.4 Sodium phosphate Tricine, ph 8. Triethanolamine, ph 7.8 Tris TBS; Tris (25mM), NaCl (.15 M), ph 7.6 Tris (25mM), Glycine (192mM), ph 8. Chelating agents EDTA 1M 2mM 25mM 25mM 25mM undiluted 1:3 dilution 1mM EGTA Sodium citrate Reducing & ThiolContaining Agents Nacetylglucosamine in PBS, ph 7.2 Ascorbic acid Cysteine Dithioerythritol (DTE) Dithiothreitol (DTT) Glucose Melibiose 2Mercaptoethanol Potassium thiocyanate Thimerosal 2mM 1mM 1mM 1mM 1mM.1% 3.M.1% Brij35 Brij56, Brij 58 CHAPS, CHAPSO Deoxycholic acid Octyl βglucoside Nonidet P 4 (NP4) Octyl βthioglucopyranoside SDS Span 2 Triton X1 Triton X114, X35, X45 Tween2, Tween6, Tween8 Zwittergent 314 Misc. Reagents & Solvents Acetone Acetonitrile Aprotinin DMF, DMSO DMSO Ethanol Glycerol (Fresh) Hydrazides Hydrides (Na 2 BH 4 or NaCNBH 3 ) Hydrochloric Acid Leupeptin 1.% 1.% 1.% 1.% 1% 1% 1mg/L 1% 1% 1% 1% 1mg/L Energenesis Biomedical Co., LTD. 6F.3, No.21, Ln. 583, Ruiguang Rd., Neihu Dist., Taipei City 114, Taiwan (R.O.C.) Website: Tel: MADE IN TAIWAN Page 6/6