What is blank in BCA assay?
Blank is used to remove the absorbance produced by the solvent in which your sample is present so that you obtain absorbance of your sample only.
What factors affect BCA assay?
Quantification and Analysis of Proteins
The development of color in the BCA assay depends on time, temperature, and pH. The assay can be performed at room (ambient) temperature, but increases the reaction temperature can significantly reduces the time required for maximal color development and increases sensitivity.
Is BCA assay linear?
The BCA/copper complex is water soluble and exhibits a strong linear absorbance at 562 nm with increasing protein concentrations.
What does a BCA assay do?
BCA (Bicinchoninic Acid) Protein Assay. The BCA protein assay is used for quantitation of total protein in a sample. The principle of this method is that proteins can reduce Cu+2 to Cu+1 in an alkaline solution (the biuret reaction) and result in a purple color formation by bicinchoninic acid.
Do you subtract blank in Bradford assay?
By convention, proteins are diluted in distilled water for the Bradford assay. Put 160 microliters water in well A-1, which will be taken to be the blank, and subtracted from the absorbances of all other wells.
What is BSA standard curve?
A standard curve was developed using a series of Bovine Serum Albumin (BSA) standards in the 100 μg/ml to 1,500 μg/ml range. The absorbance of each sample was measured at 595 nm and plotted versus [BSA]. The resulting line was fit by the linear least squared method.
Does EDTA interfere with BCA assay?
EDTA and reducing agents, such as DTT, BME, TCEP and other disulfide reducing agents, interfere with the BCA assay. The concentration of proteins can be also overestimated if proteins form soluble aggregates in solutions.
Why we use BSA as standard for protein estimation?
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.
What is the difference between BCA and Bradford assay?
The key difference between BCA and Bradford assay is that BCA assay is time-consuming and less accurate, whereas Bradford assay is quick and accurate. BCA and Bradford assay are two assay methods of protein concentration determination.
How do you do BCA protein estimation?
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.
How long does it take to do a BCA assay?
Choose the right BCA protein assay for your sample
| Rapid Gold BCA Protein Assay | Micro BCA Protein Assay | |
|---|---|---|
| Incubation time and temperature | 5 min at RT | 60 min at 60°C |
| Total assay time | 50 min | 130 min |
| Absorbance | 480 nm | 562 nm |
| Compatible reagents | Detergents | Detergents |
What is the function of the blank in spectrophotometry?
Spectrophotometers are also calibrated by using a “blank” solution that we prepare containing all of the components of the solution to be analyzed except for the one compound we are testing for so that the instrument can zero out these background readings and only report values for the compound of interest.
Why is 595 nm used in Bradford assay?
The difference between the two dye forms is greatest at 595 nm, making it the optimal wavelength to measure the blue color from the Coomassie dye–protein complex. If desired, the blue color can be measured at any wavelength between 575 nm and 615 nm.
Why is BSA used as standard?
How do you make a standard BSA curve?
To create a standard curve using your BSA standards, pipette 20 μL of each standard into an eppendorf tube. Add 980 µL of the prepared dye reagent to each tube and vortex briefly. Incubate the tubes at room temperature for 5 to 50 minutes.
Does SDS affect BCA assay?
The 2-mercaptoethanol will interfere with the BCA assay, and the SDS will interfere with the Bradford assay.
Does DTT affect BCA assay?
Some substances, including most reducing agents, interfere at even low concentrations in the BCA Assay. Examples include 5 mM DTT (for the BCA and Micro BCA™ Assays) and 0.1 M Glycine (for the Micro BCA Assay).
How many kDa is BSA?
66 kDa
BSA is a 585 amino acids, 66 kDa globular protein comprised of at least 67% α-helical secondary structure in physiological conditions [15].
Why is BSA a blocking agent?
Abstract. BSA blocking is a routine practice among clinicians and researchers working on immunoassays throughout the world. The primary role of BSA is to prevent the non-specific binding by blocking the leftover spaces over solid surface after immobilization of a capture biomolecule.
Why is BCA better than Bradford?
Historically, the BCA method is more sensitive than the Bradford method, because the first method is based on protein-copper chelation and secondary detection of the reduced copper. Whereas the Bradford method is based on protein-dye binding and colour shift from 465 to 595 nm.
What are the advantages of BCA assay compared to other methods of protein quantification?
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.
How long can I incubate BCA assay?
30-minute
The Standard Protocol permits either a 30-minute incubation at 37°C or a 2 hour-incubation at room temperature. Figure 1. BCA Enhanced Protocol assay was performed on the Modulus using the Absorbance Module and the 560 nm filter paddle.
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.
- Reagent B: 0.4 gm cupric sulfate (5 x hydrated) in 10 ml distilled water.
Why do we use blank solution?
A blank solution is a solution containing little to no analyte of interest, usually used to calibrate instruments such as a colorimeter.
What happens if you dont blank the spectrophotometer?
that absorbs light (the “chromophore”) is dissolved. Having the blank will make it possible for you to adjust the instrument so that it ignores any light absorbed by the solvent and measures only the light absorbed by the chromophore.