Hi,
Can anyone help in suggesting a method/s to compare the levels of three different proteins with each other (i.e., if one is comparing proteins A,B,C and needs to show that protein A is predominant than B and C in brain).
I know western blotting is not ideal as different antibodies have affinities. RT-PCR might be a way, but again mRNA levels doesn't necessarily correlate with protein levels.
Thanks!!
Immunoblotting would work if you can obtain pure or recombinant forms of your target proteins to 'calibrate' your IBs with. You just need to optimize your conditions such that your signals are all within the linear range of detection.
Hi there,
If the three proteins exhibit different MW (so that the 3 might be resolved by SDS/PAGE) then epitope tagging of the 3 with the same tag and use WB with anti tag probing might work.
If you have access to purified versions of the proteins for standards, and specific antibodies for each one, you could measure the actual amount of each protein in the sample using ELISAs. Slot blots can also be used quantitatively with standards. Western blot can be used as long as the standards are on the same blot as the sample.
An alternative to immunological methods is quantitative mass spectrometry. Again, standards would be needed to control for the different detection sensitivities for each protein.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies