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the protein band disappear in the second dimension


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#1 Poteo

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Posted 02 March 2005 - 09:57 PM

Hi, everybody
I tried to used the 2D-Gel for determination the puritied protein. The frist dimension was isoelectric focusing, then SDS-PAGE were analyzed in the second dimension. I stained the gel by CBB and then detaining by water:methanol:acetic acid = 8:1:1. I could not saw the protein band. If somebody have some recommentation, please tell me


Thank you very much for all recommentation.

#2 dan_

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Posted 07 March 2005 - 11:51 AM

Hi,

I have not done a lot of 2D gel, but if the protein band doesn't appears, it may be because that your protein is to much 'diluted' into the gel. Have you tried to stain your gel with something more sensitive? (like silver or Sypro (bio-rad)). Have you tried to load more protein on the gel? With a good sample, you can load easily 200-250 ug of protein.

Hope that it will help you!

#3 mabusheh

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Posted 15 March 2005 - 07:57 PM

Hi
I have done hundreds of 2-D gels, it always work fine.
I use sypro stain(bio rad) and western blot.
you have to load more protein and check your first dimension IEF. if the strip is not focused very well, you will not see bands. Also, some strips work better than others, (i.e. 3-10 and 4-7) 7 cm strips are the best,
another thing, is the Rehydration buffer you use, the composition of urea and Chaps and ampholyte are very critical in focusing the strip.
oh one more thing, I use 10% methanol+ 7% Acetic acid in Milli Q water to stain my gels with Sypro. you have to fix the gel first for at least 30 minutes then stain for 2 hours finally, destain for 2 hours.
hope this will work

good luck

Edited by mabusheh, 15 March 2005 - 08:01 PM.





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