What are the steps of BCA protein assay?
The formation of the bicinchoninic acid (BCA)–copper complex for the BCA total protein assay. This assay proceeds in two steps: the first being the reduction of Cu2 + by antibodies in a basic environment and the second step involves the reduced Cu+ chelating with two molecules of bicinchoninic acid.
What type of assay is the BCA method?
colorimetric assay
3.2 Bicinchoninic Acid Protein Assay. Bicinchoninic acid (BCA) assay or Smith assay is a copper-based colorimetric assay for total protein quantification. BCA rely on the formation of a Cu2 +–protein complex in a basic environment, followed by reduction of the Cu2 + to Cu+ (Smith et al., 1985).
What is the difference between BCA and Micro BCA assay?
While both assays use BCA (bicinchoninic acid) to measure protein concentration of dilute protein samples, the Micro BCA assay is designed for testing proteins in lower sample concentrations of 0.5-20 ug/ml, while the standard BCA assay works in the range of 20-2000 ug/ml.
Why is BCA assay the best?
One of the biggest benefits of this method is that the BCA protein assay is compatible with most protein samples and protein samples that contain up to 5% surfactants (detergents). In addition, the BCA Assay responds more uniformly to different proteins than the Bradford method.
How can I do BCA analysis?
Assay
- Prepare samples containing 0.2 to 50 micrograms protein in microliters.
- Add 1 ml SWR to each 20 microliters sample and mix. Incubate 30 min. at 60 degrees C.
- Cool the samples and read at 562 nm. Color will be stable for at least one hour.
What are the 2 reagents used in BCA?
Reagents. Reagent A: 1 gm sodium bicinchoninate (BCA), 2 gm sodium carbonate, 0.16 gm sodium tartrate, 0.4 gm NaOH, and 0.95 gm sodium bicarbonate, brought to 100 ml with distilled water. Adjust the pH to 11.25 with 10 M NaOH. Reagent B: 0.4 gm cupric sulfate (5 x hydrated) in 10 ml distilled water.
Is BCA assay accurate?
It uses about the same amount of protein as the Lowry assay, gives fairly accurate results, and is the fastest and easiest to perform among the protein assays. It is also compatible with most salts, solvents, buffers, thiols, reducing substances and metal chelating agents.
Why is BSA used in BCA assay?
The intensity of the color formed by these assays is measured by absorbance photometry at 595 nm and 562 nm for the Bradford and BCA assays, respectively. Typically, standard solutions of bovine serum albumin (BSA) are used to produce a calibration curve of absorbance versus mass concentration.
Why is the BCA method used for the protein quantitative analysis?
The BCA assay has a lot of advantages. Compared to other methods, the BCA assay is one of the most sensitive (it can detect proteins at concentrations as low as 5 ug/mL). It has less variability than others (i.e., Bradford assay), and it can be used to measure a wide range of protein concentration.
What is the absorbance in BCA assay?
562 nm
The BCA/copper complex is water soluble and exhibits a strong linear absorbance at 562 nm with increasing protein concentrations. The complex is approximately 100 times more sensitive (lower limit of detection) than the pale blue color of the first reaction.
Is SDS compatible with BCA assay?
The 2-mercaptoethanol will interfere with the BCA assay, and the SDS will interfere with the Bradford assay.
How is BCA assay calculated?
Y= mX+C (eg Y=0.0545X+0.218). Here X is the unknown concentration which you need to find. Y value is the absorbance that you got for your unknown sample. Therefore X= (Y-C)/m. i.e X= (Y-0.218)/0.0545.
How accurate is BCA assay?
The BCA assay is not perfect. Here are some limitations that you may need to consider. The reaction may be less sensitive to the type of amino acids present in the solution but the reaction is influenced by cysteine, tyrosine and tryptophan residues.
What is the purpose of protein assay?
The purpose of the protein assay is to determine the amount or concentration of a specific protein or an array of different proteins in a sample. Isolating and detecting protein is used for many clinical and research processes.
What is the wavelength of BCA?
The BCA/copper complex is water soluble and exhibits a strong linear absorbance at 562 nm with increasing protein concentrations.
What reagents are used in BCA?
Reagents. Reagent A: 1 gm sodium bicinchoninate (BCA), 2 gm sodium carbonate, 0.16 gm sodium tartrate, 0.4 gm NaOH, and 0.95 gm sodium bicarbonate, brought to 100 ml with distilled water. Adjust the pH to 11.25 with 10 M NaOH.
Why is BSA used as a standard?
BSA is used because of its ability to increase signal in assays, its lack of effect in many biochemical reactions, and its low cost, since large quantities of it can be readily purified from bovine blood, a byproduct of the cattle industry.
How do you prepare BSA standards?
Weigh 100 mg BSA and dissolve in 10 mL water to make 10 mg/mL BSA solution as stock.
What is the first step for protein assay?
The first step involves formation of a copper-protein complex in alkaline solution. This complex then reduces a phosphomolybdic-phosphotungstate reagent to yield an intense blue color. This assay is much more sensitive than the biuret method but is also more time consuming.