Disclaimer: I have run many SDS-PAGE/Western blots before and this has happened to me twice in the last couple of days.
I lysed less than 60000 cells in 70ul RIPA buffer, sonicated the samples and added 5x loading buffer with 1:100 bME, boiled for 5 minutes. I used a 10% bis-tris gel, pre-cast from Invitrogen (we've always used this before), standard MOPS running buffer, 130V, 25ul per lane, nothing different from previous runs I've done before. However the last two gels I've run looks like the photo I've attached.
I'm stuck on the problem here. My first guess was that the stacking gel somehow didn't work, but how come the ladder marker is separated? Also, it looked as if some of the samples WERE stacked properly. This doesn't look like overloading to me and I'm almost 100% sure there aren't that may proteins in the cell lysates as I've done SDS-PAGE on the same cells at much higher cell numbers lysed in lower amounts of lysis buffer.
Maybe someone here can shed some light on how to avoid this again?
I'm agree with Ruben suggestions. I'm using Bis-Tris 4-12% nupage gels with 1X MOPS running buffer at 200V 50min with my whole cell lysate in RIPA plus 2X loading buffer for Nupage (stock 4X). I strongly recommend you use this loading buffer. As Ruben did mention you should not boil samples, just 70-75 degrees for 10min is largely enough.This is the reason why you ladder is running nicer, due that you did not boil it. I have never used bMe, only DTT 50mM final concentration (from invitrogen or water resuspended DTT from SIGMA) . A couple of additional comments about these gels. Be aware about protein ladder in Nupage gels, since in catalog their sizes in kDa normally are shown on normal SDS-PAGE. I observed in your picture that you are using 1,5mm gels, just take in account that protein transfer are quite more difficult rather than 1,0mm gels. Good Luck!
Check your pH of the running buffer. I have gotten this effect for pH differences in running buffer.
Hi Joan. Be aware that NuPAGE Bis-Tris gels are quite different from traditional Laemmli SDS-PAGE. You should follow Invitrogen protocol as the Loading buffer is bit different from Laemmli loading buffer. Also Invitrogen recommends to use DTT instead bME, boil the samples at 72 oC instead 95oC, and use the antioxidant for running the gel ( I have try to do it without it and I recommend you not to forget it). If you follow Invitrogen protocol step by step your WB will be really nice. I heve try even to elaborate my own Bis-tris gels and they are really nice if you follow the protocol from Invitrogen. However two think that you should always be aware... pH of MOPS/MES running buffer and DNA presence in your lysates.
I'm agree with Ruben suggestions. I'm using Bis-Tris 4-12% nupage gels with 1X MOPS running buffer at 200V 50min with my whole cell lysate in RIPA plus 2X loading buffer for Nupage (stock 4X). I strongly recommend you use this loading buffer. As Ruben did mention you should not boil samples, just 70-75 degrees for 10min is largely enough.This is the reason why you ladder is running nicer, due that you did not boil it. I have never used bMe, only DTT 50mM final concentration (from invitrogen or water resuspended DTT from SIGMA) . A couple of additional comments about these gels. Be aware about protein ladder in Nupage gels, since in catalog their sizes in kDa normally are shown on normal SDS-PAGE. I observed in your picture that you are using 1,5mm gels, just take in account that protein transfer are quite more difficult rather than 1,0mm gels. Good Luck!
Hi joan
I got the same problem with anyKD BioRad Precast gel. After close inspection of gel, I found the gel was spoiled, having minute empty spaces in it.
I got replacement and run the gel at 80v (lower) in a ice water..it worked perfectly. I got good bands with different proteins ranging from 14-90kDa. I loaded lower amount of protein (10 ug). I hope this information will help you.
Looks like you're a year past the expiration date on the gel (06Apr13). The buffer systems (hence pH and gradients) break down over time and they generally don't run very reliably after you've gone a couple/few months past the expiration date printed on the package.
thanks guys for the tips! I saw some empty spaces (air bubbles?) in the bottom of one of the remaining gels so maybe that's causing the problem, although I'll definitely be looking into using different temperatures for boiling and loading buffers.
just to clarify, the gel was not expired, the date says april 15...
...taking your last sentences...in my hands, most of time Nupage gels are still working after 1 year out of date...
I have most likely a similar problem, but my ripa lysates have some spiky features. I thought my gel was old but got the same results when I made a fresh gel. I thought that I am maybe not letting the resolving gel set long enough before adding the stacking or could it be the RIPA (or some of its components) are just not compatible with the SDS or the gel buffers?? I could also need some advice on this
Hi Michaelone, it almost looks like the stacking gel is not homogenously mixed and the bottom has crashed out as a reason? I've never had issues with RIPA buffer on SDS gels before so I wouldn't think it's a compatibility issue...
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