Quantifying protein using absorbance at nm Considerations for use Quantifying protein by directly measuring absorbance is fast and convenient, since no additional reagents or incubations are required. No protein standard need be prepared and the procedure does not consume the protein. The relationship of absorbance to protein concentration is linear. Because different proteins and nucleic acids have widely varying absorption characteristics there may be considerable error, especially for unknowns or protein mixtures.
By Jennifer Cable When working with proteins, one key part of any good assay is accurately determining how much protein you have. There are several ways to measure protein concentration, and each of them has its own advantages and disadvantages, so how do you know which method is right for your protein?
Absorbance at nm How it works: Aromatic residues, like tyrosine and tryptophan, absorb UV light at nm. I like to denature proteins in 6 M guanidinium. This method relies on having an accurate extinction coefficient for your protein, which depends on the number of aromatic residues. Bradford Assay How it works: The Bradford assay is a colorimetric assay based on the interaction between Coomassie brilliant blue you know, the stuff you stain your gels with and the arginine and aromatic residues in your protein.
When the dye binds to these residues, its maximum absorption shifts from nm to nm. In general, you measure the absorbance of a series of known concentrations of a standard protein, generally BSA, and create a standard curve. You then use that standard curve to calculate the concentration of your protein sample based on its absorbance.
This assay is quick, and the reagent is not affected by the presence of reducing agents, like DTT and? Also, like the absorbance at nm technique, the Bradford assay depends on the sequence of your protein.
Finally, this technique depends on comparing the absorbance of your protein to that of a standard protein. BCA Assay How it works: The BCA assay is another colorimetric assay like the Bradford assay.
The procedure is similar to that of the Bradford assay, in which you create a standard curve based on a series of known protein standards.
Because the peptide backbone is involved in the reaction, the BCA assay is less sensitive to the types of amino acids in the protein. However, the reaction is influenced by cysteine, tyrosine, and tryptophan residues. The presence of reducing agents in your buffer can interfere with the dye, but there are reducing agent-compatible dyes available.
The reaction takes some time to proceed. Also, as in the Bradford assay, you determine your protein concentration by creating a standard curve from a known, standard protein. So there you have it: Choosing the right technique for your protein will take a bit of trial and error, but having a good technique in your back pocket to accurately measure your protein concentration will save you a lot of time and energy and help you get more reproducible results.nm will only give an estimation of the protein concentration unless the extinction coefficient for a pure protein has been accurately determined (by dry weight or by amino acid analysis).
Alternatively, the absorbance in the. timberdesignmag.comTION OF PROTEIN BY LOWRY’S METHOD Aim: ESTIMATION OF PROTEINS BY BRADFORD METHOD Aim: To estimate the amount of protein in the given sample by Bradford 5. ml of the supernatant is taken and the protein concentration is estimated using Lowry’s Method.
the protein concentration. This reaction occurs only with the peptide bond and not with the amino acid side chains. Because the number of peptide bonds per given unit weight is approximately the same for all proteins, this method is generally applicable and reasonably accurate, irrespective of the composition of the protein mixture.
Determination of Protein Concentration IIntroduction ntroduction Several methods are commonly used for determination of protein concentration.
Measurement of the UV absorbance at nm is most useful for pure protein solutions. Bradford and BCA assay. Note, however, that these methods may give different results for different proteins of the same concentration. Also, different methods can yield somewhat different results for the same protein.
There is no absolute photometric protein concentration assay. Expt. BL Total Protein Estimation by Lowry’s Method Objective To determine the concentration of proteins by Lowry’s method. Reagents Required.