CHM7003A Chemical and Biochemical Analytical Methods

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Reference no: EM132797814

CHM7003A Chemical and Biochemical Analytical Methods - Queen's University Belfast

Determination of total protein using BCA Assay Kit Background

The BCA Protein Assay is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantification of total protein. This method combines the reduction of Cu2+ to Cu1+ by protein in an alkaline medium (Biuret reaction) with the highly sensitive and selective colorimetric detection of Cu1+ by bicinchoninic acid, forming a purple- coloured BCA/copper complex with absorbance maximum at 562 nm. Measurement of A562 is used to determine the concentration of protein in solutions. Protein concentrations are estimated by reference to absorbances obtained for a series of standard protein dilutions (e.g. of BSA), which are assayed alongside the unknown sample. The Micro BCA Assay uses concentrated solutions and extended incubation times for the detection of dilute protein samples.

The BCA assay involves two consecutive steps of colour development, where the final product indirectly depends on the protein concentration in the solution. The first step is the chelation of copper with protein in an alkaline environment, followed by reduction of Cu2+ to Cu1+ cations forming a light blue complex. It has been shown that cysteine, cystine, tryptophan, tyrosine, and the peptide bond are able to reduce Cu2+ to Cu1+. The amount of reduction is proportional to the protein present. In the second step of the colour development reaction, bicinchoninic acid (BCA), a highly specific chromogenic reagent for Cu1+, reacts with the reduced Cu1+ cation that was formed in step one, producing a purple complex with strong absorbance at 562 nm (Smith 1985, Wiechelman 1988). The purple-coloured reaction product results from the chelation of two molecules of BCA with one cuprous ion. The intensity of the BCA/copper purple complex is proportional to the amount of protein in the sample. Thus, measuring the intensity of the absorbance at 562 nm is proportional to protein concentration. The BCA protein assay demonstrates higher tolerances towards common interfering substances, such as non-ionic detergents and buffer salts, than the Lowry technique.

Assay Protocol

Preparation of Standards and Working Reagent

A. Preparation of Albumin (BSA) Standards

You are given a 2 mg mL-1 stock solution of BSA. Complete Table 1 with the volumes of BSA and water needed to achieve the required calibration standard concentration. 1,500 μL of each calibration standard is required.

Table 1. Preparation of BSA standards

Sample ID	Water (μL)	BSA volume (μL)			[BSA] (μg mL^-1)
A			μL of 2mg mL^-1 stock		40
B				μL of A	20
C				μL of B	10
D				μL of C	5
E				μL of D	2.5
F				μL of E	1.25
G				μL of F	0.625
H				μL of G	0 (blank)

B. Preparation of BCA Working Reagent
1. Use the following formula to calculate the total volume of working reagent required:
(number of standard samples + numbers of unknown samples) × (times of repetition) × (volume of working reagent per sample) = total volume of working reagent required. mL
Note: Each sample requires equal volume (750 μL) of BCA working reagent. You are required to complete 2 repetitions of the calibration (n=3). You are required to measure total protein in 3 unknown samples.
2. Mix Micro BCA Reagent A, Micro BCA Reagent B and Micro BCA Reagent C in the ratio of 25:24:1. i.e., mix 5 mL of Micro BCA Reagent A and 4.8 mL Micro BCA Reagent B with 0.2 mL of Micro BCA Reagent C.
Note: When Micro BCA Reagent C is initially added into Micro BCA Reagent A and Micro BCA Reagent B, turbidity occurs that quickly disappears upon mixing to yield a clear green solution.

Assay Procedure

1. Add 750 μL of BCA working reagent to each standard and unknown vial and mix well. In the case of standard sample G, first withdraw and discard 750 μL of the sample and then add 750 μL of BCA working reagent.
2. Seal vials and incubate at 60°C for 1 hour.
3. Cool all vials to room temperature (RT).
4. Set the wavelength of spectrophotometer at 562 nm. Calibrate the instrument to zero by using water. Subsequently, measure the absorbance of all samples within 10 minutes.

Note: Colour development continues even after cooling to RT. However, the subsequent development at RT is too weak to produce significant error if all absorbance measurements are made within 10 minutes.

Report
1. Complete Table 1 and calculate the total volume of BCA working reagent required for this experiment.

2. Using the Absorbance data supplied, plot the BSA calibration curve: absorbance (on y axis) vs BSA Standard concentration (on x axis). Remember to subtract the absorbance of blank from all readings. Show error bars on your calibration curve.

3. Use the calibration curve to determine the protein concentration of each unknown sample.

Attachment:- Chemical and Biochemical Analytical Methods.rar

Reference no: EM132797814

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