The common methods for protein quantification mentioned in a lot of publications are Bradford assay and BCA. Due to some technical issues, I am searching other methods for protein quantification. I have been using Nanodrop for DNA and RNA quantification for years but never used for protein. There is a method in the Nanodrop for measuring protein concentration called A280. Have anybody out there used this method for protein quantification. I have to use the protein samples for western blot, so it is necessary that I load the right amount of protein in the gel?
Dear Dr. Shaima Raji Abdul Rahiman Sirajudeen,
Nanodrop has various disadvantages namely, the machine is susceptible to contamination, as the sample readings are susceptible to foreign matter. Secondly, there is a lack of sensitivity. It is recommended not to measure samples with concentration below 10ng/mL due to possible inaccurate readings. Lastly, nanodrop is sensitive to interference from a number of compounds which also includes surfactants.
However, if you want to use the Nanopdrop, you will have to wash the detector to avoid residual protein interferance, otherwise the data may be affected. Secondly, take a few blank readings in between measurements. Lastly, use a proper solution for measuring the reference sample.
Using Nanodrop for protein quantification to be used for Western Blot should not be a problem. This is because if you have decided to load a certain amount of protein on the gel which suppose may not be accurate, even then you will be loading the same amount in all the lanes, and so you will still be able to compare across different lanes.
It all depends on the experimental set-up. If your aim is to measure the expression of the protein of interest, it should be fine. But for a more complex set-up where accuracy is required, the quantification of protein by Nanodrop measurement is not recommended.
Regards,
Malcolm Nobre
Dear Shaima Raji Abdul Rahiman Sirajudeen
Protein determination by UV-visible spectrophotometer is a convenient, fast, non-destructive method for determining protein concentrations.
What problems can there be? Firstly, there may be interfering impurities in the solution, in the case of coarse cell lysate this method is not suitable precisely because of this - in the UV region in the cell many substances are absorbed, besides there are a lot of impurities that scatter light. Therefore, colorimetric methods (Bradford Lowry, etc.) are preferred for complex mixtures. Secondly, UV absorption depends on the amino acid composition, primarily on the aromatic amino acid content. Different proteins absorb differently, e.g. a 1 mg/mL solution of BSA gives an absorbance of approximately 0.6, while alpha amylase gives an absorbance of 2.42. As you can see, the error in determining the concentration can be due to variation in amino acid composition. Therefore, it is better to use the extinction coefficient for a particular protein for correct determination (1 can be used as a rough approximation).
Third, the sensitivity of colorimetric methods is higher than A280.
In general, UV spectrophotometers are a good method, but the choice depends on the application and on the sample
I agree with Malcolm; I have used nanodrop to measure protein conc but not for WBs although that should not matter.