LIT33C 8/31/98 02:25 PM Page a Bio-Rad Protein Assay For Technical Service Call Your Local Bio-Rad Office or in the U.S.
LIT33C 8/31/98 02:25 PM Page b Table of Contents Section 1 1.1 1.2 1.3 Section 2 2.1 2.2 2.3 2.4 Introduction..................................................................... 1 Principle ..................................................................................... Product Description.................................................................... Materials Required but Not Supplied......................................... 1 3 3 Instructions...............................................
LIT33C 8/31/98 02:25 PM Page 1 Section 1 Introduction The Bio-Rad Protein Assay, based on the method of Bradford, is a simple and accurate procedure for determining concentration of solubilized protein. It involves the addition of an acidic dye to protein solution, and subsequent measurement at 595 nm with a spectrophotometer or microplate reader. Comparison to a standard curve provides a relative measurement of protein concentration. 1.
LIT33C 8/31/98 02:25 PM Page 2 Table 1. Reagents Compatible with the Bio-Rad Protein Assay When Using the Standard Procedure.* Acetate, 0.6 M Acetone Adenosine, 1 mM Amino Acids Ammonium sulfate, 1.0 M Ampholytes, 0.5% Acid pH ATP, 1 mM Barbital BES, 2.5 M Boric acid Cacodylate-Tris, 0.1 M CDTA, 0.05 M Citrate, 0.05 M Deoxycholate, 0.1% Dithiothreitol, 1 M DNA, 1 mg/ml EDTA, 0.1 M EGTA, 0.05 M Ethanol Eagle’s MEM Earle’s salt solution Formic acid, 1.
LIT33C 8/31/98 02:25 PM Page 3 1.2 Product Description Protein Assay Dye Reagent Concentrate (catalog number 500-0006) contains 450 ml of solution containing dye, phosphoric acid, and methanol. One bottle of dye reagent concentrate is sufficient for 450 assays using the standard assay procedure, or 2,250 assays using the microassay procedure.
LIT33C 8/31/98 02:25 PM Page 4 Section 2 Instructions 2.1 Reconstituting the Standard To reconstitute the lyophilized bovine gamma globulin and bovine serum abumin standards, add 20 ml of deionized water and mix until dissolved. If the standard will not be used within 60 days, it should be aliquoted and frozen at -20 °C. Note: The standards contain buffer salts required for solubilizing the protein. 2.2 Standard Procedure 1.
LIT33C 8/31/98 02:25 PM Page 5 1.0 Standard procedure (20-140 µg) O.D.595 0.8 BSA IgG 0.6 0.4 0.2 0.20 0.40 0.60 0.80 1.00 Protein (mg/ml) 1.20 1.40 Fig. 1. Typical standard curve for the Bio-Rad Protein Assay, bovine gamma globulin (standard I), bovine serum albumin (standard II). O.D.595 corrected for blank - 200-1,400 µg/ml x 0.1 ml = 20-140 µg protein. 2.3 Microassay Procedure 1.
LIT33C 8/31/98 02:25 PM Page 6 0.6 0.5 Microassay procedure (1-20 µg) O.D.595 0.4 IgG BSA 0.3 0.2 0.1 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25 Protein (µg,ml) Fig. 2. Typical standard curve for the Bio-Rad Protein Microassay (1-20 µg/ml), bovine gamma globulin (standard I), bovine serum albumin (standard II). O.D.595 corrected for blank. 1.25-25 µg/ml x 0.8 ml = 1-20 µg protein. 2.
LIT33C 8/31/98 02:25 PM Page 7 3. Pipet 10 µl of each standard and sample solution into separate microtiter plate wells. 4. Add 200 µl of diluted dye reagent to each well. Mix the sample and reagent thoroughly using a microplate mixer. Alternatively, use a multi-channel pipet to dispense the reagent. Depress the plunger repeatedly to mix the sample and reagent in the well. Replace with clean tips and add reagent to the next set of wells. 5. Incubate at room temperature for at least 5 minutes.
LIT33C 8/31/98 02:25 PM Page 8 Section 3 Common Questions 1. The buffer that I normally use is not listed in the reagent compatibility list. How will I know if it interferes with the Bio-Rad Protein Assay? 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 concentration versus absorbance. If the buffer does not interfere, the two graphs of the standard curve will have identical slope.
LIT33C 8/31/98 02:25 PM Page 9 4. My sample is a mixture of proteins. Which protein standard should I use to generate the standard curve? 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.
LIT33C 8/31/98 02:25 PM Page 10 Table 2. Comparison of Bio-Rad, Lowry, and Biuret Protein Assays for 23 Proteins All assays were performed on appropriate dilutions of gravimetrically prepared 10 mg/ml solutions of commercially obtained proteins. Standards were as described in the text. Assay Results (Protein mg/ml) Biuret Lowry Bio-Rad 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 5.8 6.8 9.7 8.8 7.6 9.4 25.7 10.2 6.2 9.4 9.5 16.2 9.7 6.6 10.4 13.7 7.8 9.8 11.8 9.1 8.
LIT33C 8/31/98 02:25 PM Page 11 6. Is any sample preparation required? In general, no. However, the protein must be solubilized. (The sample can not be a suspension or an unfiltered homogenate.) 7. Can protein bound to a solid phase be quantitated? Yes, according to a recent reference in Analytical Biochemistry. Consult Anal. Biochem. 200, 195 (1992) for additional information. 8. What is the shelf life of the dye reagent concentrate and the standards? The Dye Reagent Concentrate is stable for 1 year.
LIT33C 8/31/98 02:25 PM Page 12 Section 4 Troubleshooting Guide 1. How can protein with a typical low dye response be assayed? Occasionally a protein will be assayed which yields exceptionally low color response to the Bio-Rad Protein Assay. One such protein is gelatin. Although the standard range (20-140 µg) is not detectable when the standard assay procedure is used, quantitation of the protein is possible when the sample-to-dye ratio is changed.
LIT33C 8/31/98 02:25 PM Page 13 Section 5 Ordering Information Catalog Number Description 500-0001 Bio-Rad Protein Assay Kit I, includes 450 ml dye reagent concentrate and lyophilized bovine gamma globulin standard 500-0002 Bio-Rad Protein Assay Kit II, includes 450 ml dye reagent concentrate and lyophilized bovine serum albumin standard 500-0006 Bio-Rad Protein Assay Dye Reagent Concentrate, 450 ml Related Materials 500-0005 Protein Standard I, bovine gamma globulin, lyophilized.
LIT33C 8/31/98 02:26 PM Page 14 Section 6 Safety Information MATERIAL SAFETY DATA SHEET I. PRODUCT IDENTIFICATION TRADE NAME: Methanol (as used in Protein Assay Kits I and II and Dye Reagent Concentrate) Catalog No.: 500-0001, 500-0002, 500-0006 Chemical identity, Common names: Methanol; Methyl alcohol; Carbinol; Wood alcohol; Wood naphtha, Methyl hydroxide. Formula: CH3OH M.W.: 32.
LIT33C 8/31/98 02:26 PM Page 15 III. PHYSICAL/CHEMICAL CHARACTERISTICS BOILING POINT: 64.5 ˚C (148 ˚F) VAPOR PRESSURE: 97 mm Hg at 20 ˚C EVAPORATION RATE (BUTYL ACETATE = 1): 5.9 SOLUBILITY IN WATER: Complete APPEARANCE AND COLOR: Clear liquid, alcohol odor. SPECIFIC GRAVITY(H2O = 1): 0.79 MELTING POINT: -98 °C (-144 ˚F) VAPOR DENSITY(AIR = 1): 1.1 IV.
LIT33C 8/31/98 02:26 PM Page 16 ABSORBED THROUGH SKIN: May be absorbed through intact skin and produce systemic effects. SWALLOWED: Can cause gastrointestinal irritation, headache, nausea, vomiting, blindness, unconsciousness, and death. HEALTH EFFECTS OR RISKS FROM EXPOSURE ACUTE: Poison! Swallowing or breathing high concentrations of methanol may produce headache, vomiting, nausea, irritation, dizziness, weakness, fatigue, giddiness, blurred vision, unconsciousness, blindness, and death.
LIT33C 8/31/98 02:26 PM Page 17 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 Those individuals with diseases of the eyes, liver, kidneys, and skin may be at increased risk from exposure. VI. REACTIVITY DATA STABLE X UNSTABLE_________ CONDITIONS TO AVOID: Heat, sparks, and open flame.
LIT33C 8/31/98 02:26 PM Page 18 VIII. SPECIAL HANDLING INFORMATION VENTILATION AND ENGINEERING CONTROLS: Provide sufficient mechanical explosion-proof ventilation to maintain exposure below exposure limits. RESPIRATORY CONTROLS: Wear a NIOSH-approved respirator appropriate for the vapor concentration at the point of use. Appropriate respirators may be a supplied-air respirator or a self-contained breathing apparatus. EYE PROTECTION: Chemical splash goggles or face shield.
LIT33C 8/31/98 02:26 PM Page 19 MATERIAL SAFETY DATA SHEET I. PRODUCT IDENTIFICATION TRADE NAME: Phosphoric Acid (as used in Protein Assay Kits I & II and the Dye Reagent Concentrate). Catalog No.: 500-000l, 500-0002, 500-0006 Chemical identity, Common names: Phosphoric acid, ortho-phosphoric acid, white phosphoric acid. Formula: H3PO4 M.W.: 98.
LIT33C 8/31/98 02:26 PM Page 20 III. PHYSICAL/CHEMICAL CHARACTERISTICS BOILING POINT: l58 ˚C (3l6 ˚F) VAPOR PRESSURE: 0.03 mm Hg at 20 ˚C EVAPORATION RATE (BUTYL ACETATE = 1): N/A SOLUBILITY IN WATER: Complete APPEARANCE AND COLOR: Clear, colorless, syrupy liquid. Odorless. SPECIFIC GRAVITY(H2O = 1): l.69 MELTING POINT: 2l ˚C (70 ˚F) VAPOR DENSITY(AIR = 1): 3.4 IV.
LIT33C 8/31/98 02:26 PM Page 21 SWALLOWED: Corrosive; may cause sore throat, abdominal pain, nausea, and severe burns. HEALTH EFFECTS OR RISKS FROM EXPOSURE ACUTE: Phosphoric acid is extremely corrosive to the eyes, skin, nose, mouth, throat, and mucous membranes. Bronchitis, pulmonary edema, and chemical pneumonitis may occur with inhalation of vapors or mists. Breathing high concentrations may result in death.
LIT33C 8/31/98 02:26 PM Page 22 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 History of respiratory or skin disease may increase risk from exposures. VI. REACTIVITY DATA STABLE x UNSTABLE_________ CONDITIONS TO AVOID: Contact with metals can form hydrogen gas and be an explosion hazard.
LIT33C 8/31/98 02:26 PM Page 23 VIII. SPECIAL HANDLING INFORMATION VENTILATION AND ENGINEERING CONTROLS: A local exhaust system is recommended to maintain levels below exposure limits. RESPIRATORY CONTROLS: Wear a NIOSH-approved respirator appropriate for the vapor or mist concentration at the point of use. Appropriate respirators may be a full facepiece air-purifying respirator equipped with high efficiency cartridges, a supplied-air respirator, or a self-contained breathing apparatus.
LIT33C 8/31/98 02:26 PM Page 24 Section 7 References 1. 2. 3. 4. 5. 6. 7. 8. Bradford, M., Anal. Biochem., 72, 248 (1976). Reisner, A. H., Nemes, P. and Bucholtz, C., Anal. Biochem., 64, 509 (1975). Fazakes de St. Groth, S. et al., Biochim. Biophys. Acta, 71, 377 (1963). Sedmack, J. J. and Grossberg, S. E., Anal. Biochem., 79, 544 (1977). Compton, S. J. and Jones, C. G., Anal. Biochem., 151, 369 (1985). Spector, T., Anal. Biochem., 86, 142 (1978). Duhamel, R. C., Meezan, E. and Brendal, K., J. Biochem.
LIT33C 8/31/98 02:26 PM Page 25 Bio-Rad Laboratories Life Science Group U.S. (800) 4BIORAD • California Ph. (510) 741-1000 • New York Ph. (516) 756-2575 • Australia Ph. 02-805-5000 • Austria Ph. (1) 877 89 01 • Belgium Ph. 09-385 55 11 • Canada Ph. (905) 712-2771 • China Ph. (01) 2046622 • Denmark Ph. 45-39 17 99 47 • France Ph. (1) 49 60 68 34 • Germany Ph. 089 31884-0 • India Ph. 91-11-4610103 • Italy Ph. 02-21609 1 • Japan Ph. 03-3534-7665 • Hong Kong Ph. 7893300 • The Netherlands Ph.
