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2d page - no band (Jul/19/2010 )

Hello
I m new in dis field. I am tryin 2 working with chick skeletal muscle proteins. I solubilize the protein in PB buffer and checked 4 d concentration with bradford.
what happen was, during my first run i got distorted bands , since i could nt handle the ipg-strip insertion on SDS gel side and ipg strip were damaged in places. during my 2nd run, i took the suggestion from manual and for easy insertion of ipg stip i started using ddH2O. I filled the top of the gel with ddH2O and inserted the strip and then remove the ddH2O by tilting and put agaros solution to seal the strip.
what happen is, since i started using ddH2O, I cant see any band any more after staining. is it happening because of the ddH2O?wht is the proper way of inserting IPG-strip?
I tried to repeat the sample i used for the first time which came up with bands and but no band during 2nd run. can it be because of the repeatetive thawing of the samples as wel?i stored my samples at -80C and thawed not more the once or twice.

i will be glad if any 1 could help me.
thnk u

-borna-

Hi borna,

First time when u ran the gel, u got distorted bands or spots, that u think is because of the broken strip. What I think is that could be coz of second dimension also. As u said that u were doing it for the first time, it requires lot of patience, so don't worry it will definitely work :) . I have heard that after taking out IPG strip from rehydration buffer, people wash it with water and then put it on PAGE. I have never done that. The only way to put strip is, you hold it with forcep from one side, just put it between the plates and let it go slowly, then press it gently to the gel, so that there should not be any space between strip and gel(I hope you understand :lol: ). I don't know why dint you get any spot second time :(coz u can store samples in -80 and if not reproducible, atleast you should get something on the gel........ i would suggest don't add water in that and try again. Hope this would help :D

-amj2-

When working with the strips, I tend to have the agarose bubbling hot, put it straight onto the top of the gel and then work quickly and get the strip in and push it on top of the gel with a metal spatula. Alternatively, I would suggest that you put running buffer (the buffer that is actuaally in the tank) on top of your gel and then place the strip on. I cant see why water would make a difference, but just try it with either the agarose or running buffer.

If you try to place the strip on top of the gel with no liquid, you can damage the 'gel' side of the strip (it can catch on the glass plate), which may explain your strange results.

-Lauren T-