Bio-Rad蛋白定量+protocol

DC Protein Assay Instruction Manual For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723) LIT448D 7/21/98 1:16 PM Page i Table of Contents Section 1 Introduction and Principle ……………………………… 1 Section 2 Product Description ……………………………………… 1 Section 3 Materials Required but Not Supplied…………………… 1 3.1 Safety Considerations …………………………………………… 2 Section 4 Reagent Compatibility …………………………………… 2 Section 5 Instructions ……………………………………………… 3 5.1 Standard Assay Protocol ………………………………………… 3 5.2 Microplate Assay Protocol ……………………………………… 3 Section 6 Response of Various Proteins …………………………… 4 Section 7 Storage …………………………………………………… 4 Section 8 Troubleshooting Guide…………………………………… 5 Section 9 Ordering Information …………………………………… 6 Section 10 Related Materials ………………………………………… 6 Section 11 References ………………………………………………… 7 Section 12 Safety Information ……………………………………… 8 LIT448D 7/21/98 1:16 PM Page i Section 1 Introduction and Principle The Bio-Rad DC Protein Assay is a colorimetric assay for protein concen- tration following detergent solubilization. The reaction is similar to the well-doc- umented Lowry1 assay, but with the following improvements: The reaction reaches 90% of its maximum color development within 15 minutes thereby saving valuable time, and the color changes not more than 5% in 1 hour or 10% in 2 hours after the addition of reagents. The assay is based on the reaction of protein with an alkaline copper tartrate solution and Folin reagent. As with the Lowry assay, there are two steps which lead to color development: The reaction between protein and copper in an alkaline medium, and the subsequent reduction of Folin reagent by the copper-treated pro- tein.1 Color development is primarily due to the amino acids tyrosine and trypto- phan, and to a lesser extent, cystine, cysteine, and histidine.1,2 Proteins effect a reduction of the Folin reagent by loss of 1, 2, or 3 oxygen atoms, thereby produc- ing one or more of several possible reduced species which have a characteristic blue color with maximum absorbance at 750 nm and minimum absorbance at 405 nm.2 Section 2 Product Description Reagent package (catalog number 500-0116) includes: 250 ml REAGENT A, an alkaline copper tartrate solution 2000 ml REAGENT B, a dilute Folin Reagent 5 ml REAGENT S (Sufficient for 500 standard assays or 10,000 microplate assays) The reagent package may be purchased as a kit with a bovine gamma glob- ulin standard (kit catalog number 500-0111) or bovine serum albumin standard (kit catalog number 500-0112). Section 3 Materials Required but Not Supplied For standard assay: 13 x 100 mm test tubes Reservoir for working reagent (size depends on amount of reagent that will be prepared) Pipets accurately delivering 100 µl, 500 µl, and 4.0 ml Graduated cylinders or pipets for reagent preparation Spectrophotometer set to 750 nm 1 LIT448D 7/21/98 1:16 PM Page 1 Vortex mixer Plastic or glass cuvettes with 1 cm path length matched to laboratory spec- trophotometer Test tube rack to hold 13 x 100 mm test tubes For microplate assay: Microtiter plates Reservoir for working reagent Pipets for reagent preparation Pipets accurately delivering 5 µl, 25 µl, and 200 µl Microplate reader set to 750 nm 3.1 Safety Considerations Eye protection and gloves should be worn while using this product. Consult MSDS at the end of this manual for additional information. Section 4 Reagent Compatibility The listed compounds were tested and found to be compatible with the Bio- Rad DC Protein Assay. In some cases, the presence of one or more of these substances will effect a change in the response of the protein to the assay reagents; therefore, the standard should always be prepared in the same buffer as the sample. 10% SDS 1% CHAPS 2% NP-40 1% Triton† X-100 1% CHAPSO 1% Thesit† 1% Tween† 20 1% Octyl glucoside 1% Brij†-35 0.2% C12E8* 0.1 M Tris, pH 8 0.5 M NaOH 0.5 M HCl 0.5 M (NH4)2SO4 0.025 M EDTA 0.05 M CaCl2 0.4 M Guanidine HCl 4 M Urea 0.05% Sodium azide 1 mM DTT (dithiothreitol) Note: The DC Protein Assay is incompatible with 2-mercaptoethanol (BME) † BRIJ and TWEEN are registered trademarks of Atlas Chemical. THESIT is a registered trademark of Desitin Arzneimittel GMBH. TRITON is a registered trademark of Rohm and Haas. *octaethyleneglycol dodecyl ether 2 LIT448D 7/21/98 1:16 PM Page 2 Section 5 Instructions 5.1 Standard Assay Protocol 1. Preparation of working reagent Add 20 µl of reagent S to each ml of reagent A that will be needed for the run. (This working reagent A' is stable for one week even though a precipitate will form after one day. If precipitate forms, warm the solution and vortex. Do not pipet the undissolved precipitate, as this will likely plug the tip of the pipet, thereby altering the volume of reagent that is added to the sample.) If samples do not contain detergent, you may omit step #1 and simply use reagent A as supplied. 2. Prepare 3 - 5 dilutions of a protein standard containing from 0.2 mg/ml to about 1.5 mg/ml protein. A standard curve should be prepared each time the assay is performed. For best results, the standards should always be prepared in the same buffer as the sample. 3. Pipet 100 µl of standards and samples into clean, dry test tubes. 4. Add 500 µl of reagent A' or A (see note from step 1) into each test tube. Vortex. 5. Add 4.0 ml reagent B into each test tube and vortex immediately. 6. After 15 minutes, absorbances can be read at 750 nm. The absorbances will be stable at least 1 hour. (See Troubleshooting Guide for recommendation on using a wavelength other than 750 nm.) 5.2 Microplate Assay Protocol 1. Preparation of working reagent Add 20 µl of reagent S to each ml of reagent A that will be needed for the run. (This working reagent A' is stable for 1 week even though a precipitate will form after 1 day. If precipitate forms, warm the solution and vortex. Do not pipet the undissolved precipitate, as this will likely plug the tip of the pipet, thereby altering the volume of reagent that is added to the sample.) If samples do not contain detergent, you may omit step #1 and simply use reagent A as supplied. 2. Prepare 3 - 5 dilutions of a protein standard containing from 0.2 mg/ml to about 1.5 mg/ml protein. A standard curve should be prepared each time the assay is performed. For best results, the standard should be prepared in the same buffer as the sample. 3. Pipet 5 µl of standards and samples into a clean, dry microtiter plate. 3 LIT448D 7/21/98 1:16 PM Page 3 4. Add 25 µl of reagent A' or reagent A (see note from step 1) into each well. 5. Add 200 µl reagent B into each well. If microplate reader has a mixing func- tion available, place plate in reader and let the plate mix for 5 seconds. If not, gently agitate the plate to mix the reagents. If bubbles form, pop them with a clean, dry pipet tip. Be careful to avoid cross-contamination of sample wells. 6. After 15 minutes, absorbances can be read at 750 nm. The absorbances will be stable for about 1 hour. (See Troubleshooting Guide for recommendation on using a wavelength other than 750 nm.) Section 6 Response of Various Proteins As with any colorimetric assay, different proteins will elicit greater or less- er color formation. The following proteins have been assayed with the protein assay. As demonstrated by the graph, there is a slight variation in color devel- opment with different proteins. Section 7 Storage Lyophilized preparations of Protein Standard I (bovine gamma globulin) and Protein Standard II (bovine serum albumin), if included, should be refrigerated upon arrival. These lyophilized preparations have a shelf life of one year at 4 °C. Rehydrated and stored at 4 °C, the protein solutions should be used within 60 days. Rehydrated and stored at -20 °C, the protein solutions should be used within 6 months. REAGENT A, REAGENT B, and REAGENT S should be stored away from direct sunlight at room temperature (25-30 °C). (Reagents A and B may also be stored in the refrigerator.) All reagents are good for 6 months from date of purchase. protein concentration, mg/ml ab so rb an ce BSA IgG Ribonuclease A Ovalbumin Conalbumin 2.01.00.0 4 LIT448D 7/21/98 1:16 PM Page 4 1. The buffer that I normally use is not listed in the reagent compatibility list. How will I know if it interferes with the assay? 2. My sample is a mixture of proteins. Which standard should I use for the standard curve? 3. Is any sample preparation required? 4. May I use a wavelength other than 750 nm? 5. May I store the reagents in the refrigerator? It is best to run two standard curves: One with protein in the same buffer as your sample and one with protein in water and then plot a graph of protein concentra- tion vs. absorbance. If the buffer does not interfere, the two graphs of the stan- dard curve will have identical slope. Partial interference can be compensated for by adding the buffer or interfering component to the standard curve for the actual protein assay. In any protein assay, the best protein to use as a standard is a purified preparation of the protein being assayed. In the absence of such an absolute reference protein, one must select another protein as a relative standard. The best relative standard to use is one which gives a color yield similar to that of the protein being assayed. Any purified protein can be selected as a reference standard if only relative pro- tein values are desired. Bio-Rad offers two standards: bovine gamma globulin (Standard I, catalog number 500-0005) and bovine serum albumin (Standard II, catalog number 500-0007). In general, no. However, the protein must be solubilized. (The sample can not be a suspension or an unfiltered homogenate.) Yes. Absorbance can be measured at 650- 750 nm. Yes, but all components must be warmed to 25-30 °C prior to use. Reagent S will develop a precipitate during cold room storage. Warming to 37 °C for 5 minutes will dissolve the precipitate. 5 Section 8 Troubleshooting Guide LIT448D 7/21/98 1:16 PM Page 5 6 Section 9 Ordering Information Catalog Number Product Description 500-0111 Bio-Rad DC Protein Assay Kit I, includes contents of Reagents Package and bovine gamma globulin standard 500-0112 Bio-Rad DC Protein Assay Kit II, includes contents of Reagents Package and bovine serum albumin standard 500-0116 Bio-Rad DC Protein Assay Reagents Package, does not include a standard Section 10 Related Materials Catalog Number Product Description 500-0001 Bio-Rad Protein Assay Kit I, 450 ml dye reagent concentrate and bovine gamma globulin standard for general use, based on Bradford method 500-0002 Bio-Rad Protein Assay Kit II, 450 ml dye reagent concentrate and bovine serum albumin standard for general use, based on Bradford method 500-0006 Bio-Rad Protein Assay Dye Reagent Concentrate, 450 ml dye reagent concentrate supplied without a standard, based on Bradford method 500-0005 Protein Standard I, bovine gamma globulin 500-0007 Protein Standard II, bovine serum albumin 223-9950 Disposable Polystyrene Cuvettes, 100 -3.5 ml cuvettes 224-0096 Costar 96 Well Flat Bottom EIA Plate, polystyrene microtiter plates, 5 per package, carton of 100 170-6601 Model 3550 Microplate Reader, 100/120 VAC 170-6602 Model 3550 Microplate Reader, 220/240 VAC 170-6621 Model 450 Microplate Reader, 100/120 VAC 170-6622 Model 450 Microplate Reader, 220/240 VAC For information on Bio-Rad’s extensive Microplate Data Analysis Systems, please call 800/4-BIORAD in the U.S., or contact your local Bio-Rad Representative. LIT448D 7/21/98 1:16 PM Page 6 Section 11 References 1. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., “Protein Measurement with the Folin Phenol Reagent,” Journal of Biological Chemistry, 193 (1951): 265- 275. 2. Peterson, Gary L., “Review of the Folin Phenol Protein Quantitation Method of Lowry, Rosebrough, Farr, and Randall,” Analytical Biochemistry, 100 (1979): 201- 220. 7 LIT448D 7/21/98 1:16 PM Page 7 Section 12 Material Safety Data Sheets I. PRODUCT IDENTIFICATION TRADE NAME: DC PROTEIN ASSAY REAGENT A Catalog No.: Used in Kits: 500-0111, 500-0112, and 500-0116 Chemical identity, Common names: SODIUM HYDROXIDE, Caustic soda, lye. Formula: NaOH, a caustic. M.W.: 40.00 MANUFACTURER’S NAME: BIO-RAD LABORATORIES LIFE SCIENCE GROUP 2000 ALFRED NOBEL DRIVE EMERGENCY PHONE No: HERCULES, CA 94547 510/232-7000 DATE PREPARED OR REVISED: 2/3/95 NAME OF PREPARER:ROY WOOD II. HAZARDOUS INGREDIENTS CHEMICAL NAMES CAS NUMBERS PERCENT EXPOSURE LIMITS IN AIR ACGIH TLV OSHA PEL OTHER Sodium Hydroxide 1310-73-2 1-5%* — 2 mg/m3 — *Aqueous solution also containing less than 1% sodium tartrate and less than .1% copper sulfate. III. PHYSICAL/CHEMICAL CHARACTERISTICS BOILING POINT: 100 °C SPECIFIC GRAVITY(H2O = 1): 1 VAPOR PRESSURE: N/A MELTING POINT: N/A VAPOR DENSITY(AIR = 1): N/A EVAPORATION RATE (BUTYL ACETATE = 1): N/A SOLUBILITY IN WATER: Infinite. APPEARANCE AND COLOR: Pale blue liquid. IV. FIRE AND EXPLOSION HAZARD DATA FLASH POINT: N/A FLAMMABLE LIMITS: N/A (METHOD USED): N/A EXTINGUISHING MEDIA: Not required. Use media suitable for surrounding materials. SPECIAL FIRE FIGHTING PROCEDURES: In the event of a fire, wear full protective clothing and NIOSH-approved self-contained breathing apparatus with full facepiece operated in the pressure demand or other positive pressure mode. UNUSUAL FIRE AND EXPLOSION HAZARDS: None expected . 8 LIT448D 7/21/98 1:16 PM Page 8 V. HEALTH HAZARD INFORMATION SYMPTOMS OF OVEREXPOSURE (for each potential route of exposure): INHALED: Corrosive! Inhalation not likely due to nature of the product. If product dries out, the inhaled sodium hydroxide dust may be fatal as a result of spasm, inflammation and edema of the larynx and bronchi, chemical pneumonitis and pulmonary edema. CONTACT WITH SKIN OR EYES: Corrosive! Extremely destructive to tissue of the skin and eyes. May cause irritation of eyes and with greater exposures, severe burns with possibility of blindness resulting. ABSORBED THROUGH SKIN: Corrosive! Destructive to skin on contact can cause irritation or severe burns and scarring with greater exposures. SWALLOWED: Corrosive! DANGER: May be fatal if swallowed. Causes severe burns. Sodium hydroxide is classified as a poison under Federal Caustic Poison Act. HEALTH EFFECTS OR RISKS FROM EXPOSURE ACUTE: Corrosive! Causes severe burns. See above. CHRONIC: Prolonged contact with dilute solutions or dust has a destructive effect upon tissue. FIRST AID: EMERGENCY PROCEDURES EYE CONTACT: Flush with large amounts of water for at least 15 minutes, lifting the upper and lower lids occasionally. Get immediate medical attention. SKIN CONTACT: Flush skin with large amounts of water for at least 15 minutes, while removing contaminated clothing and shoes. Wash clothes before reuse. Get medical attention. INHALED: Remove to fresh air. If not breathing, give artificial respiration. Get immediate medical attention. SWALLOWED: DO NOT INDUCE VOMITING! Give large quantities of water or milk if available. Never give anything by mouth to an unconscious person. Get medical attention immediately. SUSPECTED CANCER AGENT X NO: THIS PRODUCT’S INGREDIENTS ARE NOT FOUND IN THE LISTS BELOW. YES: _____FEDERAL OSHA ______NTP ______IARC MEDICAL CONDITIONS AGGRAVATED BY EXPOSURE: Persons with pre-existing skin disor- ders or eye problems or impaired respiratory function may be more susceptible to the effects of this substance. VI. REACTIVITY DATA STABLE X UNSTABLE CONDITIONS TO AVOID: NA INCOMPATIBILITY(Materials to avoid): Strong acid solutions, organic halogen compounds, and nitromethane. HAZARDOUS DECOMPOSITION PRODUCTS: Sodium oxide. HAZARDOUS POLYMERIZATION MAY OCCUR______ WILL NOT OCCUR X CONDITIONS TO AVOID: N/A 9 LIT448D 7/21/98 1:16 PM Page 9 VII. SPILL, LEAK, AND DISPOSAL PROCEDURES SPILL RESPONSE PROCEDURES: CAUTION: Caustic material. Cover spill with adsorbent and collect for solid waste disposal. PREPARING WASTES FOR DISPOSAL: Comply with all applicable federal, state, and local reg- ulations on spill reporting, waste handling, and waste disposal. VIII. SPECIAL HANDLING INFORMATION VENTILATION AND ENGINEERING CONTROLS: In general, dilution ventilation is a satisfactory health hazard control for this substance. RESPIRATORY CONTROLS: This product is a liquid and does not contain volatile organics and should not require any special respiratory controls. Respiratory controls may be required for pro- tection against other chemicals being used in the same area. EYE PROTECTION: Use chemical safety goggles. Contact lenses should not be worn when working with this material. Maintain eye wash fountain and quick-drench facilities in work area. GLOVES: Chemical resistant gloves such as neoprene. OTHER CLOTHING AND EQUIPMENT: Lab coat or apron. WORK PRACTICES, HYGIENIC PRACTICES: Use good laboratory practices. Wash hands after using and before eating. Do not eat, drink, or smoke in the work area. OTHER HANDLING AND STORAGE REQUIREMENTS: Keep in a tightly closed container to protect product quality. Store in a cool, dry, ventilated area. PROTECTIVE MEASURES DURING MAINTENANCE OF CONTAMINATED EQUIPMENT: Protective clothing, eyewear and appropriate NIOSH-approved respiratory protection should be worn. We believe that the information contained herein is current as of the date of this Material Safety Data Sheet. Since the use of this information and conditions of use of the product are not within the control of Bio-Rad, it is the user’s responsibility to handle the product under conditions of safe use. 10 LIT448D 7/21/98 1:16 PM Page 10 I. PRODUCT IDENTIFICATION TRADE NAME: DC PROTEIN ASSAY REAGENT B Catalog No.: Used in Kits: 500-0111, 500-0112, and 500-0116 Chemical identity, Common names: FOLIN REAGENT. Formula: MIXTURE M.W.: NA MANUFACTURER’S NAME: BIO-RAD LABORATORIES LIFE SCIENCE GROUP 2000 ALFRED NOBEL DRIVE EMERGENCY PHONE No: HERCULES, CA 94547 510/232-7000 DATE PREPARED OR REVISED: 2/3/95 NAME OF PREPARER:ROY WOOD II. HAZARDOUS INGREDIENTS CHEMICAL NAMES CAS NUMBERS PERCENT EXPOSURE LIMITS IN AIR ACGIH TLV OSHA PEL REAGENT B is a diluted FOLIN reagent containing less than 1% each of the following ingredients: Lithium Sulfate 10377-48-7 no data found Tungstic acid, sodium salt 10213-10-2 1 mg/m3 1 mg/m3 (TWA) Molybdic acid, sodium salt 10102-40-6 5 mg/m3 5 mg/m3 Hydrochloric acid 7647-01-0 7.5 mg/m3 7 mg/m3 Phosphoric acid 7664-38-2 1 mg/m3 1 mg/m3 III. PHYSICAL/CHEMICAL CHARACTERISTICS BOILING POINT: 100 °C (water) SPECIFIC GRAVITY(H2O = 1): 1 VAPOR PRESSURE: NA MELTING POINT: NA VAPOR DENSITY(AIR = 1): NA EVAPORATION RATE (BUTYL ACETATE = 1): NA ODOR: odorless SOLUBILITY IN WATER: infinite APPEARANCE AND COLOR: Clear liquid. IV. FIRE AND EXPLOSION HAZARD DATA FLASH POINT: NA FLAMMABLE LIMITS: NA (METHOD USED): NA EXTINGUISHING MEDIA: Nothing special required. Use media suitable for surrounding materials. SPECIAL FIRE FIGHTING PROCEDURES: For surrounding fire wear self-contained breathing apparatus and protective clothing to prevent contact with skin and eyes. UNUSUAL FIRE AND EXPLOSION HAZARDS: No special hazards known. 11 LIT448D 7/21/98 1:16 PM Page 11 V. HEALTH HAZARD INFORMATION SYMPTOMS OF OVEREXPOSURE (for each potential route of exposure): INHALED: Not expected due to nature of product. CONTACT WITH SKIN OR EYES: Would be irritating to eyes. ABSORBED THROUGH SKIN: Could be mildly irritating to skin. SWALLOWED: Large doses may cause gastrointestinal distress. HEALTH EFFECTS OR RISKS FROM EXPOSURE ACUTE: Contains dilute acids. Could be a mild irritant. The concentration of all the ingredients are less than 1% each. There is no specific information available for diluted FOLIN reagent. The diluted solution does contain dilute acids and heavy