Experimental reagents for BCA protein concentration detection

The Bicinchoninic Acid (BCA) method is an improvement on the BCA method, one of the commonly used protein concentration testing methods in the world. The principle is similar to that of the Lowery method for protein quantification, that is, in an alkaline environment, the protein and Cu2+ are complexed and the Cu2+ is reduced to Cu2+, and two molecules of BCA are chelated with a Cu+ to form a stable violet-blue complex, which has a high light absorption value at 562 nm and is proportional to the protein concentration, according to which the protein concentration can be determined.

Composition and storage:

Component Name Specification Save.

bca reagent 100ml 2-8℃cu reagent 2-8℃

BSA standard solution 5 mg ml 1 ml -20 C cryopreservation.

A 500 T microplate assay or a 50 T 2 ml cuvette assay can be performed.

1) Preparation of BCA reagents.

Reagent A, 1L: Weigh 10g of BCA (1%), 20g of Na2CO3·H2O (2%), Na2C4H4O6·2H2O (, 4g of NaOH ( Add water to 1L, adjust the pH value with NaOH or solid NaHCO3 to.

Reagent B, 50ml: take 2g of CuSO4·5H2O (4%) and add distilled water to 50ml.

BCA reagent: take 50 parts of reagent A and mix well with 1 part of reagent B. This reagent is stable for one week.

2) Standard protein solution: Weigh bovine serum albumin, dissolve it in distilled water and set the volume to 100ml, and make a 5mg ml solution. Dilute tenfold when used.

The BCA method is a measure of protein concentration, but in practice, it can cause a variety of errors.

1.Operational errors: Factors such as incorrect reagent use, incorrect agitation, precipitation carryover, and the time and temperature of the operation can all affect the accuracy of the measurement results.

2.Differences in reactivity: There may be differences in the responsiveness of different proteins to BCA reagents, which may lead to bias in some proteins exhibiting lower absorption values.

3.Interfering substances: Certain antibiotics, cholesterol, red blood cell lysates, etc., can interfere with the measurement results, resulting in measurement errors.

4.Protein molecular conformation: The spatial conformation of a protein may affect the reactivity of the BCA reagent, resulting in measurement errors.

5.Other factors: Factors such as the cleanliness of the sample, pH, ionic strength, etc., may also have an impact on the measurement results.

Therefore, when measuring protein concentration by BCA, it is necessary to pay attention to the operation specifications, control of interfering substances, and select appropriate standard curves to reduce the error.

1. According to the number of samples, prepare an appropriate amount of BCA working solution according to 50 volumes of BCA reagent A and 1 volume of BCA reagent B (50:1), and mix well. The BCA working solution is stable within 24 hours at room temperature.

2. Completely solubilize the protein standard, take 10 microliters and dilute it to 100 microliters so that the final concentration is. What solution is the protein sample in, and what solution should the standard be diluted. However, for simplicity, the standard can also be diluted with PBS or PBS.

3. Add the standard to the standard wells of the 96-well plate at 0, 1, 2, 4, 8, 12, 16, 20 μl, and add the standard dilution to 20 μl. 4. Add the appropriate volume of sample to the sample air of the 96-well plate and add the standard dilution to 20 μl. 5. Add 200 microliters of BCA working solution to each well and leave 37 for 30 minutes.

The concentration of the solution depends on the amount of substance dissolved in the solvent. Solubility.

It is the maximum amount of substance that a solvent can dissolve at a specific temperature.

The BCA method is certainly inaccurate when the protein concentration exceeds the range, and the excess range is generally beyond the highest point of the standard curve.

The diuret method was the first to determine protein concentration by colorimetry, ammonium sulfate did not interfere with color development, and Cu2+ complexed with the peptide bond of the protein, as well as tyrosine residues, to form a violet-blue complex, which had maximum absorption at 540 nm. The biuret method is often used to determine the content of protein solutions.

Is it a test after cleavage of intracellular proteins? It can be excluded according to the following points:

The ultraviolet absorption value is also high when the lysate is measured separately.

Whether cell membrane debris is brought in.

Whether the extraction concentration factor is too large.

Principle of protein quantification BCA method: In an alkaline environment, the protein complexes with Cu2+ and restores Cu2+ to Cu1+. BCA binds to Cu1+ to form a stable violet-blue complex with a high optical absorption value at 562 nm and is proportional to the protein concentration, from which the protein concentration can be determined.

1. The BCA method is greatly affected by the reaction temperature and reaction time. Reaction time, in particular, usually increases approximately every 10 minutes.

2. The BCA method will have a higher measurement result when the sample contains lipids. BCA methods cannot be used for concentrations of EDTA or glucose greater than 10 mM in protein samples.

3. The standard curve drift is large, so a separate standard curve should be prepared for each measurement.

The BCA method is definitely inaccurate when the protein concentration exceeds the range, and the excess range is generally beyond the standard curve.

The highest point. The biuret method was the first method to determine the concentration of proteins by colorimetry, ammonium thio.

Without interfering with color development, Cu2+ complexes with peptide bonds of proteins, as well as tyrosine residues, to form a violet-blue complex, which has maximum absorption at 540 nm. The biuret method is often used to determine the content of protein solutions.

The BCA method is a method used to determine the concentration of a protein based on its absorbance of a substance formed by its chemical reaction with Copper( ) ions. The following are the factors that may affect the protein concentration of the BCA assay:

1.Interference with the sample: If the sample is not cleaned enough or other impurities are present, it can interfere with the chemical reaction and lead to inaccurate assay results.

2.pH: According to the principle of the BCA method, the correct pH value is required to reduce the guess slip copper( ) ion to the copper ( ) ion. At the same time, the pH value of the sample also affects the efficiency of the reaction.

3.Reducing agent concentration: A high or low concentration of reducing agent may affect the effect of the reaction. In general, the concentration of the reducing agent should be within the appropriate range.

4.Centrifugation: After the addition of the reducing agent, the sample needs to be centrifuged to allow the pellet to settle to the bottom. If the centrifugation time is too short or the rotational speed is too low, the analysis results may be affected.

5.Timing of readings: For BCA methods, it is important to make readings after the reaction is complete. If the absorbance is not read at the right time, it can lead to biased results.

6.Other factors: Other factors may include the reagents provided by different ** manufacturers and batch changes in the opening of the tubes, temperature, etc.

This principle is also quoted from others.

I have used the BCA actual box, and it is indeed much more sensitive than Coomassie Brilliant Blue reagent. It's also easy to operate.

Introduction to the principle. The BCA (Bicinchoninic Acid) protein concentration quantification kit is an improved version of the BCA method, one of the commonly used protein concentration detection methods in the world. It is well known that divalent copper ions can be reduced to biuret reactions by proteins under alkaline conditions, and monovalent copper ions interact with the unique BCA Solution A (containing BCA) to produce a sensitive color reaction. The two molecules of BCA chelate a copper ion to form a purple reactive complex.

This water-soluble complex shows strong absorbance at 562 nm, and there is a good linear relationship between absorbance and protein concentration over a wide range, so that protein concentration can be extrapolated from absorbance values.

Features: 1 The procedure is simple, the assay can be completed in 45 minutes, which is 4 times faster and more convenient than the classic lowry method.

2 High sensitivity, the lower limit of detection concentration reaches 25 g ml, the minimum amount of detected protein is reached, and the volume of samples to be measured is 1-20 l.

3 The BCA method is not affected by chemicals such as detergents in most samples, and is compatible with up to 5% SDS, 5% Triton X-100, and 5% Tween 20, 60, and 80 in the sample.

4 Good linearity in the concentration range of 20-2000 g ml.

5.The coefficient of variation of different protein molecules is much smaller than that of Coomassie Brilliant Blue protein quantification.