IEF-Western blotting - (Sep/27/2004 )
Hello,
with 2D-DIGE, I have detected a hihgly phosporylated protein which I would like to further study via Western blotting. Normal SDS-PAGE did not give enough separation, so I would like to try 1D IEF (BioRad), followed by Blotting the gel. However, I do not know how to blot the IEF gel. Can I use the normal protocol for SDS-PAGE gels, or should I use some special protocol (since I am dealing with uncharged proteins, all at their iso-electric point).
I hope someone has a solution!
Simon
Hi,
I have the same problem. I wash the IEF-gel 3 times in 0,5l 50% methanol/1% SDS to get rid of the Triton in the gel and to "charge" the proteins with SDS. Then I blot 4h with 100V. I get some signals but they are still poor.
Do you have found any optimisation by now?
I have the same problem. I wash the IEF-gel 3 times in 0,5l 50% methanol/1% SDS to get rid of the Triton in the gel and to "charge" the proteins with SDS. Then I blot 4h with 100V. I get some signals but they are still poor.
Do you have found any optimisation by now?
You cannot do a regular SDS-PAGE transfer for an IEF gel. You need to do an acetic acid transfer in the reverse orientation.
I tried 3 different techniques, recently, to transfer from Ampholine PAGPlate to Nitrocellulose :
1 : semidry transfer using discontinuous buffer system (see protocol used for transfer from Servalyte prenets (http://www.serva.de/) ... unsuccessful
2 : tank transfer using Tris 25mM pH 9.2, Glycine 190mM, SDS 0.01%, Methanol 10%, 10V, 1Hr ... quite good
3 : tank transfer using Acetic Acid 0.7% (+/- Methanol 10%), 10V, 1hr ... good too (attention : do not forget to invert cathodic and anodic sides, since your proteins are charged +, in this case !!!)
Anyway, protocols 2 and 3 worked well for transferring the IEF marker, but still I have problems for focusing my sample (see my post)
Please let me know if you find some interesting things around,
Hope this will help !
Isabelle