IEF followed by Western blot - problems with poor blotting efficiency (May/01/2005 )
I have problems to blot my IEF-gels. They contain Triton X-100 ans I wash them 3times 10min in 0,5l 50%MetOH/1%SDS to get rid of the Triton and to "charge" the proteins with SDS. Then I blot for 4h with 100V. I get some signals but they are still poor.
Do anybody know an optimisation or different protocol?
What is your transfer buffer? are using 0.7% acetic acid as your transfer buffer?
thanks for replying.
I'm using a glycine/tris buffer with 20%MetOH as transfer buffer. What else except acetic acid is in your buffer?
I now tried to equilibrate the gel in 0,1M Tris/HCl pH8 to change the pH of the gel from pH4-6 to pH8 to get the proteins charged. The same would be possible in the other direction: to make the gel more acid...
Today I will see the results of my pH 8 gel but I'm very interested in your transfer buffer recipe.
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 !
The rceipie I have used is exactly as igeoris has described in the 3 metheod, however, I have always used 0.7% acetic acid in MiliQ water with no methanol with great results. Good Luck!