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BCA蛋白定量Protein Assay KitIntroduction and product overview:Protein quantification is often required before precessing protein samples for analysis.The Bicinchoninic Acid (BCA) Protein Assay is highly sensitive colorimetric assay that isnot affected by chemicals in the sample. The BCA Protein Assay primarily reduces Cu2+to Cu1+ by proteins in an alkaline environment followed by highly sensitive and selectivecolorimetric detection of BCA/copper complex. It is water-soluble and strongly absorbslight at 562nm in a linear fashion with increasing protein concentration.Kit contents:Biogot BCA Reagent A: 100ml, stored at room temperatureBiogot BCA Reagent B: 2ml stored at room temperatureBovine Serum Albumin Standard: 100mg(Power), Stored at 4℃.( Recommend to makestock solution(2mg/ml) and aliquot before store at 4℃ )Test Tube Procedures:A) Standard Preparation:Label 9 test tubes with A-I and prepare the standards as indicated below. The diluentused should be the same as used for the protein samples. The following dilutions aresuitable for duplicate Standard assays.Tube Bovine Serum Albumin Diluent(μl) Final Concentration(μg/ml)A 200μl from Stock 0 2,000B 120μl from Stock 40 1,500C 100μl from Tube A 100 1,000D 100μl from Tube B 100 750E 100μl from Tube C 100 500F 100μl from Tube E 100 250G 100μl from Tube F 100 125H 20 μl from Tube G 80 25I 0 100 0(Blank)B) Working Solution Preparation:1) Use following formula to determine the amount of working solution required.(Total number of standards and samples)*(Number of replicates)*(Volume ofworking solution sample)= Total volume working solution required.2) Mix fifty parts of BCA Reagent A with one part of BCA reagent B (50:1, ReagentA:B).C) Micro-plate Procedure (Sample to WR ratio = 1:20)1. Pipette 10 μl of each standard or unknown sample replicate into a micro-plate well(working range = 20-2,000 μg/ml).2. Add 200 μl of the WR to each well and mix plate thoroughly on a plate shaker for 30seconds.3. Cover plate and incubate in the water bath at 37°C for 30 minutes.4. Cool plate to RT.5. Measure the absorbency at or near 562 nm on a plate reader.6. Use the standard curve to calculate the protein concentration of each unknownsample.Notes1) Certain substances including reducing potential, chelating agents, and strong acids orbases are known to interfere with protein estimation and avoid those substances inthe sample buffer. (For example, EDTA, EGTA, DTT)2) Prepare a clear and fresh WR reagent at room temperature when prepping newexperiments. After adding WR reagent, it could be incubated for sixty minutes at37°C or 2 hours at room temperature. Absorbance at 562nm increases with theincreasing incubation time. Color development runs faster with the increasingtemperature. If sample concentration is too low, it will be better to run the reaction ata higher temperature or increase the incubation time.3) Good linear range for samples is from 50-2000μg/ml.4) BCA assay is interfered with by chelating agents and high concentration reducingagents. Make sure EDTA<10mM, no EGTA, DTT<1mM and β-ME <1mMin in thesample buffer. Try to remove the interfering substance by dialysis or gel filtration toeliminate or minimize the effects of interfering substances. If interference can not beovercome, it is recommend you use the Bradford protein assay kit.5) period of validity is 6 months.
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