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Product Introduction:

Under alkaline conditions, Cu²⁺ is reduced to Cu⁺, Cu⁺ forms a purple-blue complex with BCA reagent. The absorption value of Cu⁺ at 562nm is determined and compared with the standard curve to calculate the concentration of the protein to be measured. The common concentration of descaling agent SDS, Triton X-100, Tween did not affect the detection results, but was affected by chelating agents (EDTA, EGTA), reducing agents (DTT, mercaptoethanol) and lipids. In the experiment, if the background value of the sample diluent or lysate itself is found to be high, the Bradford protein concentration determination kit can be used.

Operation Instructions:

I. Microporous enzyme labeling

1. Preparation of working liquid: According to the standard product and the number of samples, add 50 volumes of BCA reagent to 1 volume of Cu reagent (50:1) to prepare BCA working liquid, and mix well (there may be turbidity when mixing, but it will disappear after mixing). BCA working fluid stable at room temperature for 24 hours.

2. Dilution standard: Take 10μL BSA standard and dilute it with PBS to 100μL (the sample can generally be diluted with PBS), so that the final concentration is 0.5mg/mL. The standard product was added to the protein standard product hole of the 96-well plate by 0, 2, 4, 6, 8, 12, 16, 20μL, and PBS was added to make up to 20μL.

3. Dilute the sample appropriately (it is best to do several gradients, such as 2x, 4x, 8x dilution), and add 20μL to the sample hole of the 96-well plate. Due to the large error of the pipette when taking a small sample, the point in front of the standard line may not be very accurate, so as far as possible, let the sample point fall behind the standard line 1/2.

4. Add 200μL BCA working liquid to each hole and place at 37℃ for 15-30 minutes. The protein concentration of A562nm was determined by enzymoleter and calculated according to standard curve. When incubating in a warm tank, care should be taken to prevent water evaporation from affecting the test results.

2. Spectrophotometer If there is no enzyme label instrument, the spectrophotometer can be mixed in the centrifuge tube and added to the colorimetric dish for color comparison. The steps are as follows:

1. Preparation of working liquid: According to the standard product and the number of samples, add 50 volumes of BCA reagent to 1 volume of Cu reagent (50:1) to prepare BCA working liquid, and mix well (there may be turbidity when mixing, but it will disappear after mixing). BCA working fluid stable at room temperature for 24 hours.

2. Dilution standard: Take 100μL BSA standard and dilute it to 1mL with PBS (the sample can generally be diluted with PBS), so that the final concentration is 0.5mg/mL.

3. Take eight (or more) 5mL centrifuge tubes, mark them, and add the reagent according to the table below.

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Set aside at 4.37℃ for 15-30 minutes. The absorption value at 562nm was measured by spectrophotometer, and the protein concentration was calculated according to the standard curve.

Note:

1. When not used for a long time, Cu reagent and PBS diluent can be stored at 2-8℃, and should be discarded if bacterial contamination is found. When the BCA reagent is crystallized and precipitated at low temperature, it can be completely dissolved at 37℃ without affecting the use.

2. If the sample contains more interfering substances, please use the Bradford protein concentration detection kit.

3. For your safety and health, please wear a lab coat and disposable gloves.

Note：Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.