While studying expression of a single protein from different cell sources by western blot technique how to calculate changes in the expression of protein by normalization?
Hey,
First of all you should also use an antibody against a housekeeping gene which should be expressed in the same amount in the different cells.
If you analyze your blot with Odysee or with another computer based program you can just select the band and the intensity will be written.
After that you just normalize to your housekeeping gene and compare the different cells.
I hope that helped and was the question.
all the best,
Sebastian
Thank you Sebastian, I understood well. I will try to work out this first then i''ll post my queries regarding this. Thank you.
Hello,
It is not always easy. Estimation of the relative amount of the proteins in cellular lysates using a housekeeping protein (actin, tubulin, etc) as a standard might be a good choice. However, if you are going to estimate the absolute amount of the protein or released protein in cell supernatant you may use other standard. Related recombinant protein (produced in bacteria and recognized by your serum) or a related commercial protein with a known concentration, or even a peptide with an epitope recognized by the antibody might be used for calibration. Peptides, for the simplicity of transfer, should not be less than 25 residues long.
Yes that is true.
You can also use a standard. But I think for westernblot it is more common to use the relative amounts.
Absolut levels can be seen more often in ELISA-experiments.
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