what is the purpose of using bsa in the bradford assay

Bovine serum albumin (also known as BSA or "Fraction V") is a serum albumin protein isolated from cows. BSA is also commonly used to determine the quantity of other proteins, by comparing an unknown quantity of protein to known amounts of BSA in, for example, the Bradford Protein Assay

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Full Answer

Should I use BSA or Bradford assay for my protein?

Join ResearchGate to ask questions, get input, and advance your work. BSA is often used for the reasons already given, but it is not always the best choice for your particular protein. Each protein responds a little differently in the Bradford assay, depending on its composition.

What are the advantages of Bradford assay?

Using Bradford can be advantageous against these molecules because they are compatible to each other and will not interfere. The linear graph acquired from the assay (absorbance versus protein concentration in μg/mL) can be easily extrapolated to determine the concentration of proteins by using the slope of the line. It is a sensitive technique.

What is BSA used for in biology?

The Bovine Serum Albumin Standard Bovine serum albumin (BSA) is a globular protein that is often used as a protein concentration standard in lab experiments as well as in numerous other biochemical applications. Consequently, why is BSA used for blocking?

What is the best way to dissolve BSA for Bradford Test?

Some use PBS to dissolve BSA while some suggest to use protein extraction buffer (the same buffer that was used to extract protein from sample) to dissolve BSA. Which is the best way to dissolve and prepare 1mg/ml BSA for standard curve in Bradford test?

Why is BSA used in assays?

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 purpose of a BSA standard?

Bovine Serum Albumin (BSA) is the standard reference for total protein quantitation by colorimetric assays. Expedeon BSA standards are designed for serial dilution to generate accurate standard curves and are precisely formulated at 2mg/ml.

Is BSA a good standard for Bradford assay?

BSA is the most commonly used standard for relative protein concentration determination in most laboratories, although the color response of γ-globulin is usually more representative of true concentration for samples that do not have a high albumin content.

Why is BSA used as a standard for protein content determination?

Generally, bovine serum albumin (BSA) works well for a protein standard because it is widely available in high purity and relatively inexpensive.

What does BSA do to cells?

In cell culture, it acts as a small molecule carrier. Because of its negative charge, Bovine Serum Albumin: Binds water, salts, fatty acids, vitamins and hormones and carries these bound components between tissues and cells.

How do you make BSA standards for Bradford assay?

Bradford Assay Materials: BSA standard solution (0.1 µg/µl) • Bradford solution o Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol. Add 100 ml of 85% phosphoric acid while stirring continuously. When the dye has dissolved, dilute to 1 l in H2O.

Why is BSA used for blocking?

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.

How does a BSA assay work?

The Bradford Protein Assay measures protein concentration in a sample. This assay works by measuring the color change achieved with the basic amino acids combined with Coomassie dye, which, under acidic conditions, changes the color of the sample from brown to blue.

What is BSA standard solution?

These bovine serum albumin (BSA) solutions are protein concentration reference standards for use in BCA, Bradford and other protein assay protocols. BSA is the universally accepted reference protein for total protein quantitation.

What is BSA concentration?

The concentration of BSA in solution can be determined by substituting the molecular weight, extinction coefficient and λ max into a derived form of the Beer - Lambert Law. A substance's λmax is the wavelength at which it experiences the strongest absorbance. For BSA, this wavelength is 280 nm.

How does Bradford assay determine protein concentration?

The Bradford assay is a quick and fairly sensitive method for measuring the concentrations of proteins. It is based on the shift in absorbance maximum of Coomassie Brilliant Blue G-250 dye from 465 to 595 nm following binding to denatured proteins in solution.

What is BSA and why is it also referred to as fraction V?

BSA is often referred to as "Fraction V", based on the original fractionation of human plasma developed by Edwin Cohn. Primarily developed for human albumin production, the process was later adapted to bovine serum albumin.

What is the Bradford protein assay?

Technically the Bradford Protein Assay is only measuring the basic amino acids, arginine, lysine, and histidine. However, most proteins have a fairly balanced level of these amino acids with all other amino acids, so we can still extrapolate the level of total protein in the sample. But, if a sample is particularly high or low in any ...

How to make a BSA reagent?

Step 1: Prepare several dilutions of the BSA standard, at least 5. For example, the dilutions may be 5, 10, 25, 50, 75, and 100 micrograms of BSA per milliliter. Step 2: Add reagent (which contains an acid and the Coomassie dye) to the BSA dilutions. Step 3: Incubate for 5 min to 1 hour.

What is the color of the Bradford assay?

Coomassie Brilliant Blue G-250, the binding dye for the Bradford Method. The Bradford assay, a colorimetric protein assay, is based on an absorbance shift of the dye Coomassie Brilliant Blue G-250. The Coomassie Brilliant Blue G-250 dye exists in three forms: anionic (blue), neutral (green), and cationic (red).

When was the Bradford protein assay developed?

For other uses, see Bradford (disambiguation). The Bradford protein assay was developed by Marion M. Bradford in 1976. It is a quick and accurate spectroscopic analytical procedure used to measure the concentration of protein in a solution. The reaction is dependent on the amino acid composition of the measured proteins.

What dye is used to bind to proteins in the Bradford method?

The Coomassie Blue G250 dye used to bind to the proteins in the original Bradford method readily binds to arginine and lysine groups of proteins. This is a disadvantage because the preference of the dye to bind to these amino acids can result in a varied response of the assay between different proteins.

What is the absorption spectrum of anionic bound dye?

The anionic bound form of the dye which is held together by hydrophobic and ionic interactions, has an absorption spectrum maximum historically held to be at 595 nm. The increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the amount (concentration) of protein present in the sample.

How long does Bradford protein assay stay at room temperature?

The dye reagent is a stable ready to use product prepared in phosphoric acid. It can remain at room temperature for up to 2 weeks before it starts to degrade.

What are the components of a protein assay?

Protein samples usually contain salts, solvents, buffers, preservatives, reducing agents and metal chelating agents. These molecules are frequently used for solubilizing and stabilizing proteins. Other protein assay like BCA and Lowry are ineffective because molecules like reducing agents interfere with the assay.

Can SDS interfere with Bradford assay?

Basic conditions and detergents, such as SDS, can interfere with the dye's ability to bind to the protein through its side chains. However, there are some detergent-compatible Bradford reagents. The Bradford assay depends on the sequence of the protein.

What is Bradford protein assay?

The Bradford protein assay is a dye-binding assay based on the differential color change of a dye in response to various concentrations of protein. The dye reagents are commonly purchased from Bio-Rad (Richmond, CA). In some research applications, this assay is recommended as a replacement for other protein assays, especially the widely used Lowry method, for several reasons. First, the Bradford protein assay is much easier to use. It requires one reagent and 5 minutes to perform as compared to the three reagents and 30–40 minutes typical for the Lowry assay. Second, because the absorbance of the dye-protein complex is relatively stable, the Bradford assay does not require the critical timing necessary for the Lowry assay. Third, the Bradford assay is not affected by many of the compounds that limit the application of the Lowry assay.

How long does it take to perform a Bradford protein assay?

First, the Bradford protein assay is much easier to use. It requires one reagent and 5 minutes to perform as compared to the three reagents and 30–40 minutes typical for the Lowry assay.

Overview

Using data obtained to find concentration of unknown

Principle

Advantages

Disadvantages

Sample Bradford procedure

Alternative assays

Further reading