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why protein and marker are not running down the gel
Posted by: abetik (IP Hidden, New member, 2)
Date: July 5, 2005 12:51PM
While I am very new at this, following the same instructions as we've done in the past, for some reason my proteins and even the marker are not even running down the gel - I've tried making a new Run buffer (same recipe as we have always used), and I tried without a stacking gel (resolving gel only) and neither are working. the fact that the marker is not even running, suggests it is not a problem with my proteins, I assume.
I noticed the mAmps is 4-8, which seems very low to me, but is the same for all the run buffers I have tried. I have not found much info on what the current should be, and cannot remember from previous attempts. I have tried 100V and 150V (for 90min each). I've also tried with different gel tanks, so do not think it is the problem. So, I need some help. Firstly, should the current be higher than it is? Could that explain why the protein marker is not running down? What might explain such a low current? Here is our Run Buffer: 0.025M Trizma Base 0.192 M glycine 0.1% SDS powder (detergent) Sodium dodecyl sulfate dd H2O Although we have been successful in the past, could one of these chemicals have gone bad? How would I know? I've re-made so many of the solutions that I can think anymore how to troubleshoot. Please help! We have run successful SDS-PAGE and Westerns following the exact same protocol and buffers, so why all of a sudden it is not running down the gel, we have no idea. thanks so much, Andrew.
Re: why protein and marker are not running down the gel
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: July 5, 2005 10:35PM
It looks like there is no currency between anode and cathode. Did you see any bubbles comeing up from bottom? It's usually a good sign of circuit. If you didn't see any tiny bubbles coming up, try double check buffers, electrophresis aparatus and power supply. If you recently switched to any brand of precast gels, be sure you have peered off the label at the lower part of the gel so that the gel can directly contact both cathod and anode buffers. Good luck.
Re: why protein and marker are not running down the gel
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: July 7, 2005 03:01PM
i agree with mitolab that it seems there is no current between the anode and the cathode. since you have tried more than one gel tank, is it possible that something is wrong with your power supply itself? check for bubbles, you should be able to see them at 150V or higher if there is a current. i've had chemicals go bad before (trizma for instance) but always had a current...my samples ran down the gel- just badly with lateral spreading.
Re: why protein and marker are not running down the gel
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: July 7, 2005 03:03PM
what is your gel formulation? if the gel tank and power supply are okay, perhaps there is something within the gel itself that is preventing an adequate current
Re: why protein and marker are not running down the gel
Posted by: abetik (IP Hidden, New member, 2)
Date: July 13, 2005 11:31AM
Thank you all who have contributed, but I have found the problem. This is a bit embarrassing, but I thought I would sacrifice the ego and share it here JUST IN CASE this could happen to someone else. When I loaded the gel (glass plates) into their frame, I loaded the lower plates facing the outside and not the inside of the tank. This of course did not provide a separation b/w upper and lower wells (or a very weak one) which explains the horrible current I was getting despite 100V. This is a silly mistake, my only consolation is that I am very new at this, and this was only the 3rd or 4th time I've done this (first one without my assistant in the country!) and well, I made a mistake. I should have been questioning things when I felt loading the markers and proteins was so easy (because the access to the wells was facing me, rather than inside the tank). If this ever helps just one person... it will be worthwhile. thanks again,
AB
Re: why protein and marker are not running down the gel
Posted by: shane (IP Hidden, Junior member, 10)
Date: August 12, 2005 12:08PM
wow that seems to be a very nice observation, and then realization.
Re: why protein and marker are not running down the gel
Posted by: Kiran (IP Hidden, New member, 1)
Date: August 16, 2005 02:08AM
This problem may be addressed in many ways. Firstly check out the PH of the acrylamide you are using. Its PH should be 7 or even less. Check out for the PH of running buffer. The recommended is 8.3. more over, prepare all the buffers fresh. Initially run it in a lower voltage i.e., at 50V, later after the proteins passes the stacking,,increase it to 100v. And lastly let me know the percent of the gel u are using I think it will help u better good luck Kiran Velpula Kiran kumar Velpula
Re: why protein and marker are not running down the gel
Posted by: olivialei (IP Hidden, Junior member, 13)
Date: August 16, 2005 09:58PM
Maybe you should try the contant current instead of contant voltage. We use 30 mA for one gel.
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