Hi,
I'm having trouble with my SDS-PAGE - some of my samples are not running, and getting caught in the stacking gel or early in the resolving gel.
Some background: I am used to loading adult mouse brain P2 membranes into 7.5% acrylamide gels, but I am afraid the protein I am currently studying may be lost in the first pellet. To study that, I decided to load three lanes of every sample on my gels: the whole-brain homogenate (whole brain extracted and homogenized in 10 volumes 1% Triton X100/PBS/Complete protease inhibitor, frozen in -80 until use), and the first-spin (13.2 krpm x 30 min at 2C) pellets and supernatants of the whole brain homogenate. The whole-brain samples are prepared on ice with 1x sample buffer/DTT, then heated at 90C for 3 mins, then sonticated and centrifuged for 5 mins to reduce bubbling. The pellet and supernatants are adjusted to approximately the same protein concentration by adding 1% Triton X100/Complete protease inhibitor/PBS, then added to 1x sample buffer/DTT on ice. They are then also heated to 90C for 3 minutes, sonticated and certifuged for 5 minutes.
So to get to the point: All my supernatant samples are running beautifully, along with my pellet and whole-brain samples for my younger ages (this is a developmental project). However, at older ages, the whole brain samples and (more particularly) the pellet samples are getting stuck at the top of either the resolving or stacking gels (or at the interface), producing a big glob at the top of my Western blots instead of a nice band in line with the others. I've tried heating the pellet and whole brain samples longer, and made sure to sonticate them very well to break up the DNA and membrane debris, but I'm still getting the blob problem. I can usually tell when it's going to occur because the sample (when loading) is very globby, sticking to the gel-loading pipet and not settling well in the well. Does anyone have any ideas for breaking up these samples more, whether through chemical or mechanical means?
I tried to upload my blot images, but couldn't manage it - it kept saying "You did not select a file to upload" after a couple minutes of saying "Uploading files". Oh well, if you want it let me know and I'll try again.
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