Hi every body. If someone is working on the 2 Dimensional gel electrophoresis (2D) USING tissue sample, i have some question regarding focusing step. the focusing result of pH 4-7 give clear spots while with pH 6-9, spots are mostly blurred not clear. Also the region of pH6-7(in pH 4-7 IPG strip) showing no spots while their are in abundance of spots in the reaming gel. similarly the region pH rangining 9/8-9 in ( in pH 6-9 IPG strip) is also blank with no spots while the reamining gel is full of spots..Your worthy suggestions will be greatly appreciated.
The basic region is always more difficult to focuse. I think you use a standard protocol with destreak or other chemicals that improve focalisation ?
Maybe you should as well be careful with the quality of strip (storage and limit date) and the quality of sample (no salt, etc...).
The IEF program has to be adapted as well for basic region, in order to limit the passive rehydratation step, to limit acrylamid degradation during too long steps...
(to find more details, see my last publication in Proteome science)
Hi,
It could be also salts in your sample. If you have too much residual salts in your sample (maybe coming from washing steps of your tissue with PBS), you will have bad focalisation of spots at the ends of your strip, particularly at basic end, making horizontal lines instead of clear spots in 2D images. Before IEF, try to precipitate your sample (TCA/acetone for example) to remove salts.
thankx laot Dinesh babu for support.
One more thing, ph 4-7 gel spotting is appropriately good, but 6-9 gel give much blurred like spotting, streaks,,?
this is also because of sample or some problem in focusing step,, my focusing condition include 200v/1hr, 500v/1hr, 1000v/1hr, 8000v/30min, 8000v/ 30 min, 8000v/5-6hr
also if IPG Buffer available from the company is for pH 6-11, and if i use the same buffer for 6-9 ipg bio rad strip, would this will make any problem?
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