Dear Colleagues, I am trying to assess the protein expression change within a same sample for multiple (four) iso-forms of that have very close molecular weight, I have tried W.B multiple times but unable to get encouragingly good results for more than two isoforms, while qRT-PCR shows variations, Please suggest how can I perform W.B for more profound expression within same sample (The molecular weight of the proteins is very very close and higher than 100kda)
Have you calculated the non-native isolelectric points of the respective proteins? 2-D gelelectrophoresis should be able to separate them If the pI differes only slightly.
You can try mass spectrometry, HPLC-ESI-TOF can discriminate several Da of difference.
Dear Ali Mahmoudpour, I have already tried the method you recommended,
still can not obtain more than 2 bands.
is there any other method of experimentation for the detection of protein expression change analyses ?
Dear Jaime Guillén, thank you very much for your such a valuable suggestion, unfortunately at the moment we can not perform HPLC-ESI-TOF presently,
Please suggest some other methods too.
Have you calculated the non-native isolelectric points of the respective proteins? 2-D gelelectrophoresis should be able to separate them If the pI differes only slightly.
Can you try non-denaturing (no SDS) native gel electrophoresis? After that, you would have to incubate in 50 mm Tris pH 7.4 with 1% SDS before transferring to membrane for the western blotting step.
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