Fat bands on 15% SDS-PAGE gel - (Mar/19/2011 )

Hi, another problem I've been having with my gels. I'm pouring a 15% resolving gel w/ a 5% stacking, using the recipe out of the molecular cloning lab manual, and I have been getting rather fat bands in the middle of the gel. I'm running a minigel in the protean tetracell, running at 50V to get through the stacking gel, then 120 through the resolving gel. After about an hour, the 10 kD marker has reached the middle of the gel, but the marker and the bands at that size are fat (like, tall and spread out, not nice concise bands). I've been allowing the resolving gel to polymerize for about an hour before pouring on the stacking, and then I let that polymerize for about an hour too. Any ideas about what could be causing this would be very much appreciated, thanks!

JesseC on Sat Mar 19 20:07:04 2011 said:

all the bands are fat or only those in the middle of the gel (middle/centre lanes?) perhaps if you post a picture, we can see what you meant...and a few more details like sample prep, loading buffer, amount you load etc....and welcome to the forum, Jesse C

Hi casandra, thanks for the response. The higher MW bands are still quite tight, only around maybe the 25 kD marker they start to broaden, getting fattest at the bottom. By fat, I mean vertically fat. Horizontally, they are still staying nicely in the shape of the lane, but they spread vertically, giving very fat bands. I'm lysing with RIPA, spin down, take the sup, do a BCA assay, mix with 6x sample buffer, boiling 7 min at 100C, then loading 20ug protein per lane. The marker and all the samples are having this problem. My boss thought that perhaps the proteins weren't stacking properly before entering the resolving gel, so I increased the size of the stacking area and run it at 50V before it reaches the resolving area, then turn it up to 120V. However, I still have the band problem. I will say that the leftover gel solution that I have in my falcon tube still has a small amount of liquid that isn't gelled after a very long time, more than 2 hours. Is that common, or is that indicative of incomplete polymerization? Thanks again for any advice here.

Oh also I've been overlaying my resolving gel with water during the polymerization period, and it was suggested to me to use isobutanol instead.

My first red flag is that your gel does not polymerize after 2 hours. Good gel should polymerize in 15'. 30'-1hour usually signs something is off in the ingredients, 2 hours is BAD. Seems like the problem is it's not polymerizing and/or pH problem. You might want to change ingredient in your gel in this order of importance:

- Polyacrylamide - make sure this is still good. If you are making 30% stock then make sure whoever make this do it correctly. We had a problem like this due to someone making too low concentration of polyacrylamide causing every gels to not polymerize.
- APS: if you freeze thaw APS more than once it'll go bad. This is the primary cause for bad gel that doesn't polymerize in an hour. Two times freeze thaw is max after that it'll go bad. Check that you don't use APS that has been Freeze/thaw for uncountable times.
- Check your Tris buffers if they have correct pH. Similar pH will cause your gel to not stack properly. Usually stacking pH is 6.8, resolving is 8.8.

- Does the fat band appear only at your marker, or it appear to other protein lanes? It might be just your marker is bad.
Last: Temed - make sure this is also still good. People usually buy TEMED from companies so there is usually no problem except if someone leave this at room temp for long time

Well the gel does polymerize in about 30 minutes, its just that the leftover stuff in the tube mostly polymerizes also, just a very small amount remains fluid for a long time. I do know that our TEMED is kept at room temp, should it be stored at 4C? I'll try borrowing some from another lab to test to see if that's the issue. Thanks

JesseC on Sun Mar 20 12:38:37 2011 said:

so you've got diffuse bands, eh and during the run, you see an ugly diffuse dye front too? your gels were not heating up? I run my 15% gels at 100 v but I don't think 120v would make a lot of difference unless there's something wrong with your running buffer etc. Like MPBM suggested- check that your buffers have the correct pH..we also keep our TEMED at 4 degrees but I know labs which don't. Prepare fresh APS. Try changing the power pac too...could be an uneven electric field. If your markers run the same way then there's probably nothing the matter with your sample. How many times have you run your gels with the same results?

i think that a component of your ripa is disrupting that part of your gel. try running the standards in a gel alone (don't add ripa to the standards, if you have been).

if you still see the fat bands then it could be that your sds has gone bad. try making fresh buffers with a different, newer, lot of sds.

if that doesn't solve the problem then try running a gradient gel. that should sharpen up the banding.

JesseC on Sun Mar 20 12:38:37 2011 said:

Oh sorry I didn't read carefully. If it polymerize in 30' then the APS and polyacrylamide should be ok.

- So yeah definitely check your pH.
- One thing to do is open one of your gel and check how hard it is (e.g. 5% is kinda soft, 15% is HARD)
- Also I forgot but check your buffer that it contains the correct 1x Tris Glycine. I've got people running stuff in WATER and/or 0.1x Tris Glycine and it was horrible.
- Make sure your inside of your gel container (cathode buffer) does not leak to outside (anode buffer) over time, because if it leaks, especially if it leaks through the top, the current will only run unevenly through the part where it's touching the gel. I think this has made my gel to run unevenly before and had smudging bands.
- What I would do is just make everything new (and do it yourself so you're sure it's fine)

- Did you cook the marker in 98C for like 5 minutes before? Usually this is what is suggested if you buy it from companies.

On 8-15% SDS gel I always ran my gel (small) at max current and 120V and it gave great result. Sometimes I run at 140V and it also give similarly great result. I've tried 160V to make it fast and it's still ok but I think it started to give smudges (and it felt really hot too)

mdfenko on Mon Mar 21 16:22:37 2011 said:

Do you think that the buffer which contain detergent (igepal?) might've disturbed the gel?