Only a few of E.coli proteins can be distinguished while most are lost. Could anyone tell me the reason why I got these results?
first, you have to add more information about your 2D protocoll, i.e. how did you perform your sample preparation, what kind of lysis-buffer, type of IPG-strips used, rehydration, sample application, etc.. To me, it seems like you have got some inpurities in your sample.
Focusing seem to be ok since the few spots on your gel are focused well.
I agree with Murat, we need more information to really help you!
However, if I look at this gel, I would say it is a pH 3-10 IPG strip. The gel % (2nd dimension) would also be helpful!. Is it a 10% gel?
If yes, I can advise you to run pH 4-7 strips, as most of your proteins will be in this range! Moreover, 12% gel should be more interesting!
The "lines" on your gel can come from salts and detergent impurities in your sample preparation!
Thank you, Murat & Pauline. Here is the protocol.
Washing cells twice with PBS Buffer. Cell pellets were resuspended in Lysis Buffer(5M urea,2M thiourea, 4% CHAPS, 65mM DTT, 20mM Tris, cocktail).After sonication for 10 min on ice , the cell debris were removed by centrifugation at 15000g for 30 min.
IPG-strips pH 3-10 . 12% gel.
Hi, for 2D electrophoresis it's very important to have a pure protein sample, especially when you have bacterial lysate. IEF is very sensitive for any impurities in your sample. Before IEF, I would precipitate your sample to remove any salts and contaminants (I usually use TCA/cold aceton precipitation. Other posibility is using microspin columns - microtubes with cut-off filter (2 or 10kDa), washing with 8M urea (or 7M urea and 2M thiourea for better solubilisation) and centrifuge in 14.000xG for getting rid of salts.
For start and better resolution I would recommend to use strips 3-10 but nonlinear (NL)
With bacterial samples it is also important to remove cell wall fragments that will interfere with focusing (you tend to see this manifest as a streak at ~pH 4, as you seem to have in your gel). The easiest way to remove this is to run your sample through a high MWCO filter (I use 300 kDa cutoff) and check the conductivity before loading the strips.
hi
i think there is a problem of streaking in ur gel. this may be because of several reason. first u have to check agarose sealing at the time of strip loading to the SDS-PAGE, increase the amount of protein alomost double. third run the IEF for up to 18000V. I hope this will improve the quality of your gel and no of protein spots..
Regards
Murli Manohar
Since you did not purify the protein, the sample most probably contains a lot of nucleic acids and other non-protein stuff which is very bad for 2-D.
Use purification kit or at least precipitate proteins (with aceton, for example, or with TCA. If you use TCA, wash well the pellet to remove acid)
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