5 replies to this topic

[microdexter]

Hello everyone, here is a problem I have been having with every 2D gel I have done recently. A gel image is attached.

I have no idea what caused the volcano-like blurring at the bottom of the gel. I know this is not caused by gel distortion because every gel has this phenomenon.

This gel was runned on Bio-rad Protean XL 17cm system. I used hand-casted 12%-20% tris-glycine gel.

Anybody has similar experience before?

Thanks a lot!

Attached Thumbnails

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Edited by microdexter, 29 September 2010 - 08:14 PM.

[mdfenko]

before or after you run the gel, do you see air trails between the plate and the gel?

the pattern i see in your picture looks like air entered at the bottom of the gel (like the plates separated).

this can be caused by the way the gel is handled when mounted to the apparatus, dirty plate(s), and/or warped plates.

Edited by mdfenko, 30 September 2010 - 07:08 AM.

[microdexter]

mdfenko, on 30 September 2010 - 07:08 AM, said:

before or after you run the gel, do you see air trails between the plate and the gel?

the pattern i see in your picture looks like air entered at the bottom of the gel (like the plates separated).

this can be caused by the way the gel is handled when mounted to the apparatus, dirty plate(s), and/or warped plates.

Hmm, I indeed saw air trail like you described, especially when the running is near the end.

Let me try to handle the gel more carefully and see if there is any improvement.

Many thanks.

[mdfenko]

other ways to cause the gel to separate from the plate include high acrylamide percentage (especially in gradient gels) and gel heating.

[microdexter]

mdfenko, on 01 October 2010 - 09:59 AM, said:

other ways to cause the gel to separate from the plate include high acrylamide percentage (especially in gradient gels) and gel heating.

The high acrylamide percentage is very likely the cause in my case because the phenomenon was much less severe when I used to use 8%-16% gels.

Well my acrylamide percentage can't go any lower, any suggestions?

[mdfenko]

microdexter, on 01 October 2010 - 03:57 PM, said:

The high acrylamide percentage is very likely the cause in my case because the phenomenon was much less severe when I used to use 8%-16% gels.

Well my acrylamide percentage can't go any lower, any suggestions?

i've worked with high percentage gels, as well.

first, make sure your plates are as clean as possible, don't give the gel a reason to pull off the plate (also, make sure they are not warped).

then, make sure the gel is completely polymerized and does not already display the air tracks.

finally, be careful when handling the plates. make sure you don't push or slide the plates apart, especially when mounting on the apparatus.

when you run the gel, don't let it heat up. if necessary, let it take longer to run.