Hi one of my projects include looking at phenotypic changes upon knocking down of a certain protein. So i started first by looking for expression levels of this protein by western blot. However i found very little expression. Using 50ug of total protein i couldnt get any signal using the normal ECL substrate but. i see some bands although not very prominent using the stronger substrate in luminogen at 1 min exposure.
Iisit still worthwhile to perform a knockdown?
Have you tried at least two distinct antibodies against your proteins by western blot to be sure of its expression level?
Did you analyse its mRNA level ?
It is always worth to perform a knockdown but you need a way to specifically quantify it, at protein and/or mRNA levels.
A "knockdown" is only successful and worthwhile doing when - (i) one can detect the mRNA and protein selectively at constitutive or basal levels reproducibly; (ii) it is possible to quantitate a decrease in the expression of mRNA and/or protein after "knockdown"; and (iii) the knockdown does not have a phenotype that results in significant cell growth inhibition.
There is a chance that the protein goes along with insoluble fraction during lysis and clarification.
- Badges
- Science method