5 replies to this topic

[barnacleman]

Please help!

In my 2D gels almost all proteins at the basic end (i use pH 3-10 strip) disappeared whereas all others on the acidic end is still present.
What would be the likely causes?

Is the equilibration time not enough?
Is the concentration of DTT too high?
Rehydration of samples into the IPG strips poorly?
Staining efficiency of Colloidal Coomassie G-250 stain is not good enough?
Or others?

Thanks in advance.

[fishdoc]

barnacleman, on May 7 2010, 11:48 PM, said:

Please help!

In my 2D gels almost all proteins at the basic end (i use pH 3-10 strip) disappeared whereas all others on the acidic end is still present.
What would be the likely causes?

Is the equilibration time not enough?
Is the concentration of DTT too high?
Rehydration of samples into the IPG strips poorly?
Staining efficiency of Colloidal Coomassie G-250 stain is not good enough?
Or others?

Thanks in advance.

Are you sure there should be proteins there?

[Prep!]

yeah actually i second that.... which is because one of my collegue went mad wen his acidic proteins started showing basic bands till he realised he was reading the gel upside down!!!  

[mdfenko]

what are you using for your basic end electrolyte? if you are using naoh then you may be decomposing the protein.

are you sure that you are using a 3-10 strip? if you use a strip that doesn't go to 10 then you may be running your basic proteins off.

staining efficiency should not be your problem but you can try silver staining to confirm.

[fishdoc]

mdfenko, on May 10 2010, 01:51 PM, said:

are you sure that you are using a 3-10 strip? if you use a strip that doesn't go to 10 then you may be running your basic proteins off.

Would the proteins run off of an IPG strip? I've wondered that since I did some 2D a few years back. The system we used had the IPG strips laying across two electrodes so that a short piece of the IPG gel laid outside the electrode on each end. Would the proteins run off the strip (past the electrode) or would they just accumulate at the point of where the electrode contacted the gel.

[mdfenko]

fishdoc, on May 10 2010, 05:39 PM, said:

Would the proteins run off of an IPG strip? I've wondered that since I did some 2D a few years back. The system we used had the IPG strips laying across two electrodes so that a short piece of the IPG gel laid outside the electrode on each end. Would the proteins run off the strip (past the electrode) or would they just accumulate at the point of where the electrode contacted the gel.

the protein would run into the electrode strip, accumulate at the electrode.

a short piece of gel past the electrode would just be excluded. you may as well just put the electrodes at the end.