To quantify the presence of a set of genes (responsible for the receptor expression) in a given cell-line, which methods are feasible and recommended?
I am sure that the quantification of protein expression can be done by Western Blot technique. What other alternative techniques would be applicable?
I look forward for your valuable recommendations and responses.
Hello Akshaya Nagarajan,
The two most common methods for protein quantification are based on either mass spectrometry or immunological techniques like Western Blotting.
Western Blotting is often used for protein identification or relative quantification. It can also be used for absolute quantification if appropriate calibration standards are used.
Mass spectrometry based techniques offer superior data quality and reproducibility. It can achieve low limits of detection, provided that technical pitfalls, such as incomplete protein extraction, incomplete proteolysis, and artifactual protein modifications, are appropriately controlled and considered.
You may use selected reaction monitoring (SRM), also referred to as multiple reaction monitoring.
A Western blotting assay essentially depends on the specificity of the antibody used. In contrast, an SRM assay depends on multiple parameters, such as the retention time, the mass-to-charge ratio of the precursor ion and selected fragment ions of the targeted peptide, and the relative signal intensities of the detected fragment (transition) signals.
These values are then weighted and combined to derive a score that indicates the probability that the targeted peptide has been detected.
The paper attached below will be helpful.
https://pubs.acs.org/doi/10.1021/pr049852e
Best.
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