western blot -- running the gel - (Apr/13/2008 )
hi guys.. i need some help
i was running two gels last night for 10.5 hrs at 10 mA, max voltage. they were both connected to the same apparatus/unit. today when i came in BOTH gels only ran half way. i'm strapped for time and i was wondering if i could continue running the gels? would that work? or should i just start again?
also, if i connect both gels to one apparatus, do i have to change the current?
any help would be extremely appreciated. =D
We keep the voltage constant when running gels (although I don't exactly know why). There was recently a discussion about it if I remember correctly.
So, if you run to gels connected to one apparatus and keep the voltage const., there won't be any problem. However, if you run them on cons current, the voltage will decrease.
I try to imagine that in a simple way that the electrons that are about go from minus to plus, create a certain tension between minus and plus. The current would be like the number of electrons going out from minus to plus. If you keep the current const and run to gels in the same apparatus, it will lead that half of electrons goes through one gel and the other half to the other gel (because you defined the number of electrons by choosing const current). The means the tension between the minus and plus pole of each gel will be reduced as only half number of electrons are passing now.
If you choose const voltage, the number of electrons has to be increased at the apparatus so that the tension can be const for each gel.
I hope that was kind of understandable??
I am sure that expert views would help more:)
I agree with Zek, I always run at constant voltage and often run 4 gels on the same power pack and I've not had a problem.
As to whether you can carry on, If you are doing a western blot, you may find that your bands have lost their resolution if you carry on, did the gel stop running? or was it running very slowly? It depends on how critical this gel is, if its really important, then start again, if its a routine one, carry on but be prepared to do the gel again. If you see a result at least you know your antibodies work and you can repeat to get a nicer looking gel.
I'm not sure of the size of your gels, but could you not make and run them again at 200V for a shorter time (mini gels 45mins-1 hour)?
Maybe that helps
Lost in the lab
when you run gels in parallel the current splits. if you have two equal gels then the current will split in half for each gel (10mA=5mA/gel). if you run at constant voltage then you don't need to worry whether the current splits.
if your gel was running slowly then it may not have gone bad (diffuse). you can increase the current and finish the run.
10.5hrs! Am I abnormal in running my gels at 150V for 1.5 hrs? Should I run them slower?
When we run big gels, we run it at 80 V until the front reaches the separating gel. Then it runs at 120V until the front runs out of the gel and maybe a bit longer if the bands need to be more separated. The whole running time is about 9 h (basically a whole day in the lab).
We run the small gels at const. 80V.
we run our "small" gels (24ml before stacking) at 5mA overnight (~16hr) then crank it up to 15mA to finish the run (400V limit).
we run our "large" gels (63ml before stacking) at 15mA overnight then finish with 30-50mA (400V limit).