Hey everyone,
while running two 2D SDS-PAGE mini gels I face the problem, that one of these gels runs much faster than the other one (sometimes the slower gel runs only 2/3, while the other one is finished). Since some of my proteins that I want to detect are fluorescently labeled, I don't really have the time to take the faster gel out and let the other one finish.
- The samples running in both gels are similar --> different (wt and resistant) cell lines treated with the same active agent, both precipitated before the 1st dimension
- The percentage of both gels is the same (8-16 %)
- The gels are both precasted and quite fresh.
I don't see this problem in other simultaneously run gels, even though they also carry samples from different cell lines. Does anybody know what could be the reason and how to get rid of the running difference, since the loss of running distance is especially annoying in 2D separation. Thanks in advance!
sounds like the platinum electrode wire in the running tank may be damaged/ bent or have some corrsion on them. Look very close at your wires, also if there is a small leak in the tank check the gaskets well too.
Dont over fill when running as well.
Best of Luck!
are they both run together on the same apparatus or on 2 similar but distinct electrophoresis rigs and are they running straight down the gel or is there any difference in the shape or direction of the slower gel image?
If fluorescence is an issue can you run the gel in a dark box or room?
sounds like the platinum electrode wire in the running tank may be damaged/ bent or have some corrsion on them. Look very close at your wires, also if there is a small leak in the tank check the gaskets well too.
Dont over fill when running as well.
Best of Luck!
Check the followings:
1. Protein level in each of the lanes (overloaded).
2. Leak out of the tank.
When i have to run two gels, i run them on separate power packs. My observation in the lab has always been that, although mini gel apparatuses can accomodate two gels only one of them runs good.
Then I think Charles is right.Check that all connections are clean and bright and that the platinum wires are clean and make good contact with the copper connectors,Wash any crystalline deposits away gently with water and check that the connectors are not loose.
Thanks so much for all your answers!
@Paul: I run them on the same apparatus. I'd say, that the slower gel is definitely more crooked than the fast one. And I always cover the whole apparatus with a cardboard box to keep out the light, but the fluorescence scanner that I use is at another institute, so I can't measure the first gel while the slower one is still running...
@Charles: I checked, but I couldn't find any noticable problems... and since the electrophoresis works just fine with other samples run simultaneously, I can't image that it's a general problem with the apparatus...
@Ayoob: The tank does not leak, I checked. Also I only have a single "lane", since my proteins are migrating out of the IPG strip into the 2nd dimension.
@Mangal: Sadly we only have this one apparatus, which is quite big for a mini gel apparatus (Cleaver Scientific). All the other ones are smaller (BioRad) and I would like to separate my proteins on the bigger mini gel apparatus in order to make sure the best separation is managed (for a mini 2D gel...) - quite complicated, I know! :)
@Hassan: Any other suggestions what could be the problem with only the one sample? That difference sadly is not a one-time wonder, but I repeatedly see these migrating differences whenever I run a gel with that "cell line combination" but not with others, which is why I am rather reluctant to blame the apparatus... Thanks in advance!
@Sunil: Yes, they share the same buffer chamber! But sadly I can't mix my sample before loading it to the gel, since all my proteins have already been separated by isoelectric focusing and are in an IPG strip, which I just put onto the precast gel (so I can't do something about gel polymerization as well)... Thanks anyways!
Today I'm switching it up a little, so that the problematic sample is on the other side of my buffer chamber, just to definitely rule out that the wires are damaged somehow... if that doesn't work, I'm slowly running out of options though.
@Sophie , I think your idea was good and my help you figure out if wires had an issue. Did you checked if your samples were completely soluble? are they protease-free ? are they pure enough?
Good luck
A few questions:
1. Do you run a pre-stained marker with your sample and does it behave the same way?
2. Can you feel any difference in temperature of the cassettes/glas plates between the two gels after the run?
3. What was the result of the side-switch?
Hey Vivica,
1. Yes, the prestained marker I use behaves the same way.
2. I don't notice a severe temperature difference of the plates between the two gels...
3. It worked out quite well... the two gels containing different samples ran the same distance this time...so the wires don't seem to be the problem... but since the "problematic sample" ran just as far when loaded onto the other side, this doesn't seem to be the problem as well... ?
Are both samples prepared in the same way ? Are you using IPG strips for the 1st dimention ? same amt of ampholites ? same amount of salts ? Next run place the gels for the 2nd dimension in the reverse location in the gel box.
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