Problem loading SDS-PAGE gel - (Oct/31/2006 )

Hello

I am a new graduate student and just started doing western blots. I tried loading today but evrytime it seems like samples are bubbling out into adjacent wells. I am told that its pippeting technique and it comes from practise.

I have read that some people use syringes to load gels and some people use the reverse pipeting to ensure accurate loading. Should I try those or should I just keep on practsing? Any tips or help will be much appreciated.

Thanks

what kind of tips are u usin?
i am using SDS PAGE long tips and its great and yes u load slowly and move ur hand up as u are loading and keep in mind that each coomb got its maximum amount of loading.

it has sth to do with experience; but there may be other reasons for your problem:

if gels are self-made there may be some acrylamide left in the slots which disturb loading, so abscise aa rests

there may also be a too low concentration of glycerol in your loading sample buffer; you should increase the amount of glycerol;

if your sample buffer + protein is more dense than running buffer, samples should flow into the slots independent of loading with syringe or pipette tips (special loading tips are superfluous as they often clog)

try gel loading tips.
try different pipets, sometime the tip is not perfectly adapted and buffer starts to enter the tip when you plunge the tip in the well, sometime pipets are too hard, and you can not pipet slowly easily. You must be comfortable.
aspirate the exact volume, so you don't have bubbles in the tip.
pipet very slowly, and be patient.

Thanks for all the replies. Nevr thught I would get so many responses for a basic question like mine.

To the question of what tipes I use, I use the long slender one. Id ont really know the name, but its supposed to be used for loading gels since it looks different than the others.

And Kosmodrom, I use premade gels. And the loading buffer ? use is Laemmli buffer and add Beta Mercaptol to it. I willtry adding some glycerol ti it next time.

And I go very slow but when it comes time to go to the second stop the bubbles come out. ALso will reverse pipeting help since I dont have to go past the first stop?

Thanks again

reverse pipetting could help. the normal way will come good with practice though

one technique my postdoc taught me is to gently touch the tip to the wall (left wall if you're right-handed and vice versa) of the well and let the sample slide SLOWLY down it into the well. it works!

Since this a Blot Post I was directed over here:
Recently I have had problems with my blots as well. I'm getting a ton of bands where I should not have nearly so many.
Blots I have done in the past have turned out well so I'm confused about this new occurence.
I receieved some new plasmids that I transfected into Cos-7 cells and subsequently made lysates and IPs from these. I ran 3 separate gels and transfers only to keep getting an enormous amount of bands especially in my lysate blot.
Thinking it might be a problem with this particular plasmid I ran a transfection of known plasmids on the same cell type, prepped lysates and IPs, ran a gel and transfer only to get the same results: So many bands I get dizzy looking at them all. The known plasmids have been used before and I've gotten clean results with them so I know what it should look like.
It seems my technique has fallen apart someplace. I had my PI hang out with me while I prepped the lysates and IPs, he could find no complaint in my following the protocol. And when I loaded the gel I had him observe to ensure there was something I wasn't missing. I don't see any leakage from the wells. I don't think I'm overloading the wells either.
I try very hard to keep everything on ice and if it must be removed it is only for a very short necessary period of time (i.e. long enough to pipet and load the sample). Has anyone had this problem before? How did you correct for it?
Amelia

what about ur antibodies?
did u change them?or change dilution?