PROTEIN QUANTIFICATION KIT - Wide Range

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1 Revised January 11, 2010 PROTEI QUATIFICATIO KIT Wide Range Technical Manual Product Code: PQ0210 (500 tests) PQ0212 (2500 tests) Contents General Information... 1 Contents of Protein Quantification Kit Wide Range... 1 Storage... 1 How to Use Protein Quantification Kit Wide Range Required equipments and materials Protocol Materials that Interfere with the assay Proteintoprotein variation... 2 otes... 2 Product Code and Price...2 Related Products... 3 General Protocol at a Glance...4 For the most uptodate technical manual, access 30 West Gude Drive, Suite 260, Rockville, Maryland Toll free: Phone: Fax: info@dojindo.com Web site:

2 GEERAL IFORMATIO For protein concentration determination, several methods, such as Lowry method, bicinchoninate method (CA method), iuret method and radford method, are available. Lowry and CA methods are the most popular methods for protein quantification. Since both Lowry and CA methods are based on the monovalent copper ion detection with monovalent copper ion specific chelating agents, a high concentration solution of divalent copper ion is required. Protein Quantification KitWide Range is based on the reducing reaction of tetrazolium salt under basic conditions. Tetrazolium salt is easily reduced with sugar residues of proteins, and generates formazan dye. WST8 formazan dye is yellow at neutral ph, and turns blue at higher ph. The maximum wavelength of the formazan dye is 650 nm at ph 12.5 or higher. The detection range of this kit is from 50 µg/ml to 5 mg/ml (SA). Since the sensitivity of the protein assay depends on the type of proteins, please note the proteintoprotein variation in quantification. + WST8 OCH 3 Figure 1. Chemical structure of WST8 and its formazan dye a) 400 a + reducing OCH 3 Figure 2. Absorption spectrum of WST8 with and without protein a) protein free b) 2000 µg SA/ml Wavelength/ nm Formazan/ basic condition COTETS OF THE KIT Wide Range b) 800 STORAGE Store the kit at 05 o C with protection from light. WST8 Solution is stable for 12 months at 05 o C and 3 months at room temperature. uffer Solution is stable for 18 months at room temperature. However, ph of uffer Solution may drop due to carbon dioxide in the atmosphere. Please close the cap tightly after use. SA Standard Solution is stable for 12 months at 05 o C. HOW TO USE THE KIT Wide Range STORAGE 1. Required Equipments and Materials Microplate reader (650 nm filter) 96 well microplates 10 µl, µl pipettes, multichannel pipette 1.5 ml tubes 2. Protocol 1) Dilute SA Standard Solution using ddh 2 O with multiple dilutions to prepare various concentrations of SA Standard solution (stock concentration of SA Standard Solution is µg/ml) µg/ml, 2500 µg/ml, 1250 µg/ml, 625 µg/ml, 313 µg/ml, 156 µg/ml, 78 µg/ml, 0 µg/ml 2) Add 180 µl uffer Solution to each well. 3) Add 20 µl of various concentrations of SA Standard solution from Step 1 or sample solution to each well, and mix. 4) Add 20 µl WST8 Solution to each well, and mix. 5) Cover the plate with an aluminum foil a) and incubate it at 37 o C for 30 min. 6) Measure the absorbance of each well at nm with a microplate reader. 7) Subtract the absorbance of blank solution from the absorbance of each well. 8) Plot the concentration of SA on the Xaxis and the absorbance value on the Yaxis to prepare a calibration curve (Fig. 2). Determine the protein concentration of unknown sample using the calibration curve. a) WST8 is light sensitive after mixing with uffer Solution, and the background of the solution may increase without the protection from light. Please protect the solution from light during incubation. WST8 Solution ml x 1 bottle uffer Solution ml x 1 bottle SA Standard Solution (10 mg/ml) ml x 1 vial 1

3 2 1.5 a) The compatible concentration (in sample solution) was determined within +5% fluctuation of the slope of SA calibration curve Proteintoprotein Variation Concentration of SA ( µ g/ml) This kit determines the protein concentration of sample solution using SA as a standard solution. Therefore, the concentration determined is not an absolute protein concentration. The proteintoprotein variation in quantification is shown in Table 2. Figure 3. Typical Calibration Curve Prepared using SA Standard Solution 3. Materials That Interfere with the Assay The maximum noninterfering concentrations of materials, which may interfere with the protein quantification using Protein Quantification Kit Wide Range, are shown in Table 1. Table 1 Compatible concentrations a) of possible interfering materials with WST8 assay Chemical Concentration Detergent rij % rij % rij % Triton X % Triton X % Tween % Tween % SDS... 1% CHAPS... 4% CHAPSO... 2% MEGA % OctylβDglucoside...0.5% Organic solvent Ethanol... 10% Isopropanol...10% DMSO... 10% Chelating agent EDTA mm DTPA mm Salt Sodium chloride M Potassium chloride... 1 M Sodium acetate M Sodium bicarbonate mm uffer Citrate ph mm MES ph mm Tris ph mm PS... no interference HEPES ph mm CHES ph mm Table 2 Proteintoprotein variation in quantification Protein Protein vs. SA a) SA Chymotrypsinogen A Transferrin Human IgG a) Values were determined by the comparison with the slope of calibration curve: value=slope of protein/ slope of SA OTES 1. Store the kit at 05 o C. 2. Since the sensitivity of Cbased protein assays depends on the type of proteins, please note the proteintoprotein variation in quantification (Table 2). For more accurate quantification, use the same protein, which you want to measure, as the standard. 3. Since the uffer Solution is highly basic, please handle with care. 4. The ph of the uffer Solution may drop due to carbon dioxide in the atmosphere. Please close the cap tightly after use. 5. WST8 is light sensitive after mixing with uffer Solution and the background of the solution may increase, so please protect from light during incubation. 6. Please confirm that excess amount of interfering materials is contained in the sample solution (see Table 1). If the amount of the interfering material is high, dilute it to reduce the concentration of interfering materials prior to use. PRODUCT CODE AD PRICE Product Unit Product Code Price ($) PQK WR* 500 tests PQ0210 $70 PQK WR* 2500 tests PQ0212 $198 * PQKWR: Protein Quantification KitWide Range 2

4 RELATED PRODUCT Product Unit Product Code Price ($) PQK*Rapid 500 tests PQ0110 $70 PQK*Rapid 2500 tests PQ0112 $198 * PQK: Protein Quantification Kit OTES For technical questions regarding this product, please contact us at (toll free) or send info@dojindo.com. U.S.A. Dojindo Molecular Technologies, Inc. 30 West Gude Dr. Rockville, MD Phone: Fax: info@dojindo.com Web site: JAPA Dojindo Laboratories Kumamoto Techno Research Park Tabaru, Mashikimachi, Kamimashikigun Kumamoto , JAPA Phone: Fax: info@dojindo.co.jp Web site: 3

5 General Protocol at a Glance Read Technical Information carefully prior to using this General Protocol Step 1) Prepare Standard SA Solution. a) 500 µl 500 µl 500 µl 500 µl µg/ml SA 500 µl ddh 2 O 500 µl a) dilute µg/ml Standard SA stock solution with ddh 2 O to prepare various concentrations of SA solutions by multiple dilutions: 0, 78, 156, 313, 625, 1250, 2500, 5000 µg/ml 1/2 1/4 1/8 1/16 Step 2) Add 180 µl of uffer Solution to each well. b) b) the use of 3 wells per one standard SA solution or sample solution (n=3) is recommended. Step 3) Add 20 µl of Standard SA Solution or sample solution to each well, and mix. Then, add 20 µl of WST8 Solution to each well, and mix. Sample Solution or Standard SA Solution WST8 Solution Step 4) Incubate the plate at 37 o C for 30 min with protection from light. c) 1 hour c) Light causes increase in background OD. Step 5) Put the plate in a microplate reader and read the O.D at nm, then determine the concentration of protein in a sample solution from the calibration curve. 4 Protein Quantification KitWide Range: PQ02

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