蛋白定量23250说明书

INSTRUCTIONS Pierce Biotechnology PO Box 117 (815) 968-0747 www.thermoscientific.com/pierce 3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax Number Description 23250 Pierce BCA Protein Assay Kit − Reducing Agent Compatible, sufficient reagents to assay 250 samples or standards of 25µL each Assay Working Range: 125-2,000µg/mL Kit Contents: BCA Reagent A, 250mL, contains sodium carbonate, sodium bicarbonate, bicinchoninic acid and sodium tartarate in 0.1M sodium hydroxide BCA Reagent B, 25mL, contains 4% cupric sulfate Compatibility Reagent, 10 × 20mg Reconstitution Buffer, 15mL Albumin Standard, 2mg/mL, 10 × 1mL ampules, contains bovine serum albumin at 2.0mg/mL in 0.9% saline and 0.05% sodium azide Storage: Upon receipt store at room temperature. Introduction The Thermo Scientific Pierce BCA Protein Assay Kit − Reducing Agent Compatible enables quantitation of total protein in samples while minimizing interference from disulfide reducing agents. The BCA assay is based on the well-known reduction of Cu+2 to Cu+1 by protein in an alkaline medium and the highly sensitive and selective colorimetric detection of the cuprous cation using bicinchoninic acid (BCA).1 Disulfide reducing agents, particularly dithiothreitol, 2-mercaptoethanol and TCEP are also capable of reducing Cu+2 to Cu+1. To minimize the affect of these copper reducers, a compatibility reagent that modifies disulfide reducing agents is added to the sample before adding the BCA reagents. This assay is also compatible with most ionic and non-ionic detergents in the presence of a disulfide reducing agent. Purification of membrane proteins presents unusual challenges to protein quantitation, as these proteins often require the presence of detergents and a disulfide reducing agent to maintain solubility and stability. The dual compatibility of this kit enables researchers to more accurately determine protein concentration for such samples. Important Product Information • The Pierce BCA Protein Assay Kit − Reducing Agent Compatible is compatible with protein samples containing up to 5mM DTT, 35mM 2-mercaptoethanol or 10mM TCEP. • Certain substances interfere with the Pierce BCA Assay Kit − Reducing Agent Compatible, including chelating agents and strong acids or bases. Please see the Interfering Substances Section for more information. • If the sample’s pH is ≤ 5.0, maintain the ionic strength of the sample buffer at ≤ 50mM. When two or more interfering substances are present in the sample (e.g., DTT and SDS), the buffer ionic strength must be ≤ 20mM. • Standard curves generated in the range of 125 to 2,000µg/mL using bovine serum albumin (BSA) or bovine gamma gobulin (BGG) with and without disulfide reducing reagents produce < 5% slope variation (see Figure 1 in the Additional Information Section). • If either BCA Reagent A or Reagent B precipitates upon shipping in cold weather or during long-term storage, dissolve precipitates by gently warming and stirring solution. Discard any kit reagent that shows discoloration or evidence of microbial contamination. 1745.4 23250 Pierce® BCA Protein Assay Kit − Reducing Agent Compatible Pierce Biotechnology PO Box 117 (815) 968-0747 www.thermoscientific.com/pierce 3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax 2 Additional Materials Required • 1.5mL microcentrifuge tubes • Pipettors and disposable pipette tips • 37°C water bath Procedure for the Reducing Agent-Compatible BCA Assay A. Blank and Standard Preparation The best method to control for the effects of reducing agent is to add the same type and amount of reducing agent to each serially diluted protein standard as occurs in the samples. However, because this method is laborious and typically yields < 5% error compared to standards prepared without added reducing agent, the default procedure uses a simpler method involving two blanks. The Standard Blank has no reducing agent, allowing background absorbance of the standards to be determined; the Sample Blank contains reducing agent, allowing background absorbance of the samples to be determined. • Standard Blank: This Standard Blank does not contain protein. Prepare 200µL of the same buffer as the unknown sample(s) without reducing agent. • Sample Blank: This Sample Blank does not contain protein. Prepare 200µL of the same buffer as the unknown sample with reducing agent at the same concentration as the sample. • Protein Standards: Dilute the contents of one Albumin Standard (BSA) ampule into several microcentrifuge tubes, preferably using the same buffer as the unknown sample(s). Use the following table as a guide to prepare a set of standards with sufficient volume for three replications (assay range = 125-2,000µg/mL): Vial Diluent Volume (µL) BSA Source and Volume (µL) Concentration (µg/mL) A 0 500 of stock 2,000 B 125 375 of stock 1,500 C 200 200 of stock 1,000 D 200 200 of vial B 750 E 200 200 of vial C 500 F 200 200 of vial E 250 G 200 200 of vial F 125 B. Reagent Preparation • Working Reconstitution Buffer: Dilute the Reconstitution Buffer 1:1 with ultrapure water. For protein samples with a buffer pH < 5, do not dilute the Reconstitution Buffer. • Compatibility Reagent Stock Solution: Add 1mL of either Working Reconstitution Buffer or Reconstitution Buffer to one tube of Compatibility Reagent and vortex at high speed for 30 seconds to dissolve. Store this solution for up to 8 hours at 4°C protected from light. • BCA Working Reagent (WR): Use the following formula to determine the total volume of WR required: (# blanks + # standards + # unknowns) × (# replicates) × (volume of WR per sample) = total volume WR required. Example: For three unknowns and two replicates of each sample: (2 blanks + 7 standards + 3 unknowns) × (2 replicates) × (1mL) = 24mL WR required. To prepare the WR, mix 50 parts BCA Reagent A with one part of BCA Reagent B (50:1, Reagent A:B). Note: When Reagent B is added to Reagent A, the solution appears turbid but yields a clear, green WR upon mixing. C. Protein Quantitation 1. Pipette 25µL of each replicate of standard, unknown sample and the Standard and Sample Blanks into 1.5mL tubes. 2. Add 25µL of Compatibility Reagent Stock Solution to each tube and vortex at low speed to mix. Proper mixing of samples with Compatibility Reagent Stock Solution is essential for assay accuracy. 3. Incubate tubes at 37°C for 15 minutes in a water bath. Using a forced-air incubator can introduce significant error from uneven heat transfer. Pierce Biotechnology PO Box 117 (815) 968-0747 www.thermoscientific.com/pierce 3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax 3 4. Add 1mL of the WR to each tube and vortex to mix well. Incubate tubes at 37°C for 30 minutes in a water bath. 5. Cool tubes at room temperature (RT) for 5-10 minutes. 6. With the spectrophotometer set to 562nm, zero the instrument on a cuvette filled only with water. Subsequently, measure the absorbance of all the samples within 10 minutes. Note: Because the BCA assay does not reach a true end point, color development will continue even after cooling to RT. However, because the rate of color development is slow at RT, no significant error is introduced if the 562nm absorbance measurements of all tubes are made within 10 minutes of each other. 7. Subtract the average 562nm absorbance value of the Standard Blank replicates from the 562nm absorbance value of all standards. 8. Subtract the average 562nm absorbance value of the Sample Blank replicates from the average 562nm absorbance value of unknown sample replicates. 9. Prepare a standard curve by plotting the average blank-corrected 562nm value for each BSA standard vs. its concentration (µg/mL). Use the standard curve to determine the protein concentration of each unknown sample. Troubleshooting Problem Possible Cause Solution No color in any tubes Sample contains a copper chelating agent at an incompatible concentration (see Table 1) Dilute, dialyze or desalt the sample Increase copper concentration in Working Reagent (e.g., use 50:2, Reagent A:B) Blank absorbance is OK, but standards and samples have less color than expected Strong acid or alkaline buffer altered the Working Reagent pH Dilute, dialyze or desalt sample Color of samples including blank appear darker than expected Sample contained reducing agent at concentration above the indicated compatible level Dilute sample Sample contains biogenic amines (catecholamines) Dilute, dialyze or desalt sample Sample contains lipids or lipoproteins Add 2% SDS to the sample to eliminate interference from lipids2 Interfering Substances Certain substances interfere with the reducing agent-compatible BCA assay, including those with reducing potential, chelating agents and strong acids or bases. The following substances interfere even at very low concentrations: ascorbic acid, catecholamines, creatinine, impure glycerol, hydrogen peroxide, hydrazides, iron, certain lipids, melibiose, phenol red, impure sucrose, tryptophan, tyrosine, uric acid. Other substances interfere to a lesser extent, and they have only minor (tolerable) effects below a certain concentration in the original sample. Maximum compatible concentrations for many substances are listed in Table 1. The listed concentrations are compatible with either 5mM DTT or 35mM 2-mercaptoethanol after modification with the Compatibility Reagent. Substances were considered compatible if the error in concentration estimation caused by the presence of the substance and the reducing agent was ≤ 10%. Blank-corrected 562nm absorbance values for 1,000µg/mL BSA and the Compatibility Reagent-modified substance were compared to the net 562nm values of the same standard prepared in water. Table 1. Compatible substance concentrations for the BCA Protein Assay Kit−Reducing Agent Compatible.§ Detergents* Buffers/Salts Chelators Tween®-20 Triton® X-114 Triton X-100 CHAPS SDS Octyl β-thioglucopyranoside Zwittergent® 3-14 10% 2% 10% 10% 10% 10% 2% Tris HEPES, pH 7.5 MES, pH 6.1 Imidazole, pH 7.0 Guanidine•HCl Urea Sucrose 50mM 200mM 100mM 50mM 2M 4M 40% EDTA EGTA Sodium citrate 20mM 10mM 100mM *Detergents were tested using high-purity Surfact-Amps Products, which have low-peroxide content. §For a more extensive list of substances, download Tech Tip # 68: Protein Assay Compatibility Table from our website. This Tech Tip includes compatible substances for all of our protein assays and enables easy comparisons. Pierce Biotechnology PO Box 117 (815) 968-0747 www.thermoscientific.com/pierce 3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax 4 Additional Information Standard curves generated in the range of 125 to 2,000µg/mL using bovine serum albumin (BSA) with and without disulfide reducing reagents produce < 5% slope variation (Figure 1). 0 0.2 0.4 0.6 0.8 1 1.2 0 500 1,000 1,500 2,000 N et A bs or ba nc e at 5 62 n m - DTT + DTT 0 0.2 0.4 0.6 0.8 1 1.2 0 500 1,000 1,500 2,000 - 2ME + 2ME 0 0.2 0.4 0.6 0.8 1 1.2 0 500 1,000 1,500 2,000 - TCEP + TCEP BSA (µg/mL) Figure 1. Standard curves generated using BSA with Compatibility Reagent and the presence and absence of DTT, 2-mercaptoethanol and TCEP. Standard stock solutions were prepared in 20mM Tris•HCl, 0.05% sodium azide, pH 8.0. Related Thermo Scientific Products 23209 Albumin Standard Ampules, 2mg/mL, 10 × 1mL ampules 23252 Pierce Microplate BCA Protein Assay Kit – Reducing Agent Compatible, sufficient reagents for 1,000 microplate assays 23208 Pre-Diluted Protein Assay Standards: BSA Set, 7 × 3.5mL ranging from 125 to 2,000µg/mL 23212 Bovine Gamma Globulin Standard, 2mg/mL, 10 × 1mL ampules 23213 Pre-Diluted Protein Assay Standards, bovine gamma globulin fraction II (BGG) Set, 7 × 3.5mL ranging from 125 to 2,000µg/mL 23221 Pierce BCA Reagent A, 1,000mL 23223 Pierce BCA Reagent A, 250mL 23224 Pierce BCA Reagent B, 25mL 23235 Pierce Micro BCA Protein Assay Kit, working range of 0.5-20µg/mL 23236 Pierce Coomassie Plus (Bradford) Assay Kit, working range is 1-1,500µg/mL References 1. Smith, P.K., et al. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem. 150(1):76-85. 2. Kessler, R. and Fanestil, D. (1986). Interference by lipids in the determination of protein using bicinchoninic acid. Anal. Biochem. 159(1):138-42. This product (“Product”) is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale, as set forth in the Product documentation, specifications and/or accompanying package inserts (“Documentation”) and to be free from defects in material and workmanship. Unless otherwise expressly authorized in writing, Products are supplied for research use only. No claim of suitability for use in applications regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This warranty does not extend to anyone other than the original purchaser of the Product (“Buyer”). No other warranties, express or implied, are granted, including without limitation, implied warranties of merchantability, fitness for any particular purpose, or non infringement. Buyer’s exclusive remedy for non-conforming Products during the warranty period is limited to replacement of or refund for the non-conforming Product(s). There is no obligation to replace Products as the result of (i) accident, disaster or event of force majeure, (ii) misuse, fault or negligence of or by Buyer, (iii) use of the Products in a manner for which they were not designed, or (iv) improper storage and handling of the Products. Current product instructions are available at www.thermoscientific.com/pierce. For a faxed copy, call 800-874-3723 or contact your local distributor. © 2011 Thermo Fisher Scientific Inc. All rights reserved. Unless otherwise indicated, all trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries. Printed in the USA. 5mM DTT 35mM 2-Mercaptoethanol 10mM TCEP Introduction Important Product Information Troubleshooting Interfering Substances Related Thermo Scientific Products References