I can see my target protein band clearly around 333 seconds but for my housekeeping protein, I can see it clearly around 10 seconds with total protein loading is 40ug. May I know do I need to standardise the exposure time of the target protein & housekeeping protein with ECL?
Yes, you need to standardize the exposure time for housekeeping protein and target protein since their expression levels may be different. Normally, housekeeping protein expression levels are relatively higher. So we also use different ECL reagents for detecting housekeeping protein (normal sensitive ECL) and the target protein (super sensitive ECL).
Hi Sir Venkateswarlu. Thank you very much for the comment. Can you elaborate in more details?
We probe the blot first using target protein antibody, advanced (super sensitive) ECL and different exposure times. After that we strip the blot and re-probe using house keeping protein antibody, normal ECL and different exposure times to make sure that we are not getting equal loading because of saturation of the signal.
It is very often the case that the target protein is expressed at a much lower level than the housekeeping gene. You should always show the house keeping gene blot as loading control along with the target gene blot in figures.
Some researchers scan the images in then use a software such as ImageJ or ImageQuant to perform densitometry to represent the target protein as a relative fold change over the housekeeping gene. This can account for variations in loading between lanes. However, this is all relative and NOT quantitative.
Best practice is to make sure neither of the blots are overexposed. Overexposed western blots can mask differences in signal and significantly affect how you interpret your results. Hope this answers your question.
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