What are the steps of BCA protein assay?
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 is BCA protein assay?
The BCA protein assay is used to quantify total protein in a biological sample. BCA stands for Bicinchoninic acid, which is the key reagent used to produce a colored product. The purple colored product is analyzed in reference to a standard curve in order to quantify protein concentration.
How does the Pierce BCA protein assay work?
The BCA Protein Assay combines the well-known reduction of Cu2+ to Cu1+ by protein in an alkaline medium with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu1+) by bicinchoninic acid (BCA).
Does SDS interfere with BCA assay?
Note: As used in this protocol, 5% SDS is compatible with the BCA Protein Assay.
How long does a BCA assay take?
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 role of copper in a BCA protein assay?
BCA is a weak acid composed of two carboxylated quinoline rings and serves the purpose of the Folin reagent in the Lowry assay, namely, to react with the product (Cu1 + ions) of complexes between copper ions and peptide bonds to produce a purple end product that strongly absorbs at 562 nm (Smith et al., 1985).
How do you dilute a BCA sample?
This is how I typically dilute my samples:
- Transfer 20 µL of each sample to a new cDNA tube/Eppendorf tubes.
- Add 60 µL of deionized water to the samples (dilute by a factor of 4).
Does urea interfere with BCA?
The BCA assay is generally tolerant of ionic and nonionic detergents such as NP-40, Triton X-100 and denaturating agents like urea and guanidinium chloride that tend to interfere with other colorimetric protein assays such as Lowry (Table 2) [15].
Which protein assay is most sensitive?
the BCA assay
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 used as the standard solution in BCA assay?
The standard Pierce BCA Protein Assay detects protein concentrations from 20 to 2,000 μg/mL using a two-component system: Reagent A, a carbonate buffer containing BCA reagent, and Reagent B, a cupric sulfate solution, which are combined to make an apple green–colored working solution that turns purple after 30 minutes …
What is the Pierce BCA assay kit?
The Thermo Scientific ™ Pierce BCA Protein Assay Kit is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein. This method combines the well-known reduction of Cu +2 to Cu +1 by protein in an alkaline medium (the
Which amino acids are involved in BCA color formation?
The macromolecular structure of protein, the number of peptide bonds and the presence of four particular amino acids (cysteine, cystine, tryptophan and tyrosine) are reported to be responsible for color formation with BCA.
How do you prepare diluted albumin standards?
A. Preparation of Diluted Albumin (BSA) Standards Use Table 1 as a guide to prepare a set of protein standards. Dilute the contents of one Albumin Standard (BSA) ampule into several clean vials, preferably using the same diluent as the sample(s).