Protein Quantification Kit (BCA Assay)

Transcription

1 Protein Quantification Kit (BCA Assay) Booklet Item NO. KTD3001 Product Name Protein Quantification Kit (BCA Assay) ATTENTION For laboratory research use only. Not for clinical or diagnostic use Version

2 TABLE OF CONTENTS INTRODUCTION principle... 1 Storage/ Stability... 1 Assay Restrictions... 1 PRODUCT INFORMATION Materials supplied... 2 Other supplies required... 2 Technical hints... 2 ASSAY PROTOCOL Reagent preparation... 3 Assay procedure... 3 DATA ANALYSIS Calculation of results... 4 Typical data... 4 Version

3 INTRODUCTION principle Abbkine Protein Quantification Kit (BCA Assay) provides a simple, rapid, detergent-compatible procedure for the colorimetric detection and quantitation of total protein. The method utilizes the reduction of the copper cation (Cu 2+ ) to the cuprous cation (Cu + ) by protein in an alkaline medium. The generated Cu + forms an intensely purple-colored complex with the bicinchoninic acid (BSA) with a very strong absorbance at 562nm. The intensity of the water-soluble complex is proportional to the amount of protein in the sample. The BCA Protein Assay is suitable for measuring protein concentration in the range of µg/ml (1-20 µg protein). Storage/ Stability Stable for at least 12 months at 4 C from date of shipment. The BCA Reagent A and BCA Reagent B are stable at 4 C. The BSA Standard should be aliquoted after the first thaw and stored at -20 C. Assay Restrictions Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted. 1

4 PRODUCT INFORMATION Materials supplied components Storage 500T 2000T 5000T conditions BCA Reagent A (2 ) 50 ml 200 ml 500ml 4 C BCA Reagent B 2 ml 8 ml 20ml 4 C BSA Standard (10 mg/ml) 1 ml 4 ml 10ml -20 C Booklet RT Other supplies required 37 C incubator. Microcentrifuge. Pipettes and pipette tips. Colorimetric microplate reader or spectrophotometer. 96 well plate or Test-tube. Orbital shaker. Technical hints To avoid cross-contamination, change pipette tips between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent. When BCA Reagent B is first added to BCA Reagent A dilution, turbidity is observed that quickly disappears upon mixing to yield a clear, green BCA Working Solution. The BCA protocol is very flexible. Both the incubation time and temperature can be varied over a rather wide range. Lower protein samples can be more easily quantified using higher temperatures and longer incubation times. Certain substances are known to interfere with the BCA assay including those with reducing potential, chelating agents, and strong acids or bases. Because they are known to interfere with protein estimation at even minute concentrations, avoid these substances as components of the sample buffer. Certain substances interfere to a lesser extent with protein estimation using the BCA assay, and these have only minor (tolerable) effects below a certain concentration in the original sample. Such as, NP-40, SDS, Triton X-100, Tween-20 and so on. When assaying protein in solutions containing detergent, best results are obtained by adding the same amount of detergent to the wells containing the protein standard. 2

5 ASSAY PROTOCOL Reagent preparation Bring all reagents to room temperature before use. If crystals have formed in the BCA Reagent A Concentrates, warm them gently until they completely dissolved. BCA Working Solution - Prepare working solution by adding 1 part of distilled water to 1 part of BCA Reagent A, then adding 1 part of BCA Reagent B to 50 parts of the above dilution. The total volume made will depend upon the number of samples and standards to be quantitated. Each sample and standard will require 200 μl of working solution. Once made, the Working Solution is stable for a week. Sample Solution - Dilute samples to fall within mg/ml range. Assay procedure A. Microplate Procedure 1. Standard Solution Preparation. Label 8 tubes 0-7. Dilute the BSA Standard to 1 mg/ml working Solution. Ideally, use the same buffer contained in your samples. Then prepare serial dilutions as follows. Tube BSA working Solution (μl) Buffer (μl) Concentration (μg/ml) Pipette 20 μl Standards or samples into duplicate wells in a clear bottom 96 well plate. 3. Add 200 μl of BCA Working Solution into each well that contains the standard or samples. 4. Shake gently to mix. Incubate for between min at C. Cool to room temperature. 5. Measure OD at 562 nm. The signal is stable for at least 30 minutes. B. Test-tube Procedure According to the specification of the Test-tube, as in the above method, the volume of each solution may be appropriately increased in proportion. 3

6 DATA ANALYSIS Calculation of results Subtract the blank OD (zero standard) from all standard and sample OD values. Plot the corrected OD against standard protein concentrations. Use the standard curve to determine the sample protein concentration. Alternatively, the equation for the best line fitting the standards can be used to determine the protein concentration of your samples. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Typical data The standard curve is provided for demonstration only. A standard curve should be generated for each set of samples assayed. OD562nm BSA Concentration in µg/ml 4