I cannot find any detailed explanation regarding this question, please help!
In blue native PAGE, almost all protocols mentioned to replace the heavy blue cathode buffer (i.e. in my case, 0.02% Coomassie blue) with light blue cathode buffer (0.002% Coomassie blue), after the dye runs til one-third of the gel.
If possible, please attach some materials for me to refer to. Many thanks in advance!
It is important for sub sequent analysis. Coomassie is negatively charged and it will block efficient protein transfer during western blotting as it will compete with proteins for binding to PVDF membrane. It also interferes with in gel fluorescence detection and enzymatic assays. We also had troubles with subsequent mass spectrometry if the dye was not removed. You do not need to exchange the buffer if you are going to use it for detection of radioactive proteins or visualization of protein complexes via coomassie staining. For a reference see paper attached.
Article High Resolution Clear Native Electrophoresis for In-Gel Func...
It is important for sub sequent analysis. Coomassie is negatively charged and it will block efficient protein transfer during western blotting as it will compete with proteins for binding to PVDF membrane. It also interferes with in gel fluorescence detection and enzymatic assays. We also had troubles with subsequent mass spectrometry if the dye was not removed. You do not need to exchange the buffer if you are going to use it for detection of radioactive proteins or visualization of protein complexes via coomassie staining. For a reference see paper attached.
Article High Resolution Clear Native Electrophoresis for In-Gel Func...
Replacing the buffer with low Coomassie cathode buffer half-way through the run effectively destains the gel. This is useful for subsequent analysis afterwards, as if your complexes are relatively abundant they can directly be visualized just by this destaining step. However, replacing the buffer is not essential, and for Western blotting, we have found that incubating the get in transfer buffer containing 0.1% SDS is sufficient to replace the Coomassie to allow efficient transfer. For more information, if you want to skip this step, please see
Article Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for...
I sometimes replaced it with buffer without any Coomassie just to better see the protein bands. You can also try to just load your samples with deep blue cathode buffer and use cathode buffer without coomassie. It worked for my samples but i was just looking for samll proteins and no membrane proteins at all..
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