Hey
I am planning to run a 1D electrophoresis, to separate proteins according to their IEF. I have the Novex invitrogen ph3-7 reagents here. What is the amount of protein I need to load into the ready-made gel?
Can I stop here and do the coomassie staining without following it up with 2D electrophoresis?
is the lysis buffer made up from Urea, chaps buffer etc..?
Thank you
Best wishes
Marie: There is no best efficient way for all cases/person. You need to optimize your system for your protein. For analytical purpose, you don't need a lot of proteins for visualization. For preparation purpose, you can load up to 100 micrograms per lane, and cut band later for MS analysis. We only do 1-D for our protein (PMID: 22949401; PMID: 25900303). Good luck.
Hi Marie,
for purified protein isoelectric point determination we have used Bio-Rad 111 Mini system loading enough amounts to be visualized under coomassie or silver staining. But if your plan is a complex protein mixture 2D separation then you should not do any staining after 1D and follow a protocol for that purpose.
Hi Marie,
1) the amount of protein that you can load on a IEF strip depends on the length of the strip, so you have to check the product data sheet to verify this point. For example, the Bio-Rad IPGs of 11 cm that I have often used, can be loaded with 20 to 200 micrograms of total protein (it depends also on which kind of staining you want to use after the second dimension, higher the sensitivity lesser the amount of protein). However I agree with Ke-Wei, every system has to be optimized.
2) yes you can stop the first dimension and stain the strip if you want to check your run
3) usually the lysis buffers are suitable for the IEF runs so you can use Urea, CHAPS etc.
I hope this will help you
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