I'm attempting to probe for low molecular weight proteins (11-25 kDa) from whole-cell lysates using a Tris based RIPA lysis buffer with inhibitors. I've been trying to separate these extracts using a 12% SDS-PAGE gel (Tris-Glycine buffer system with a 4% Stacking gel), 5x sample buffer, protein concentration 20 mg/ml and 30ul per well. However, a dye 'blob' keeps forming in each lane at the gel front. The 'blobs' become more apparent as the separation proceeds and a small purple band also forms in front of them over the course of the run at constant 100V. The size marker runs fine, other than the smallest two marker bands run off the gel before the 'blob' reaches the bottom which I'm guessing means I've also lost the proteins I'm interested in. How do I make the dye front tighten up into a single band?
9/1/18 - GOOD NEWS EVERYONE! I FIGURED OUT THE PROBLEM!! The sample buffer we were using was the issue. Originally, we were using a 5x sample buffer with beta-ME and freezing 1ml aliquots. When a fresh batch was made, DTT, instead of beta-ME, was used; this is what was producing the dual-colored dye front. The solution that we've decided on is to start with aliquots of 5x NON-REDUCING sample buffer. Upon thawing a new aliquot, 25% beta-ME is added. This has completely resolved the issue. See a new image below. Thanks to everyone who offered advice - the science community really is the BEST!
12/8/17 Okay everyone, NEW UPDATE: I've tried eliminating variables individually; here's what I've got so far:
The gel in the color picture contains NO PROTEIN. It is just 6ul 5x sample buffer (2 separate batches) & 24ul RIPA lysis buffer. Ran the gel using a colleague's Tris-glycine separating (12%) and stacking (4%) buffers. My colleague and I also share the same 1x running buffer, other reagents and equipment. (I have my own power supply but have never had an issue with it on previous runs.)
Made my own fresh Tris buffers and ran another gel with 6ul 5x sample buffer and 24ul of 1% Triton-X 0.25M 7.2pH Tris-glycine as a control against the RIPA again with NO PROTEIN. Same result for both (not pictured).
12/5/17 UPDATE: Hi everyone, thanks for your responses so far. I've attached two images. I didn't have a picture of the gels, unfortunately, but the 'blobs' transferred to the membrane, which I have pointed out. I'm wondering: I just started using RIPA for lysis (same time the blobs appeared); also, could it be caused by a higher-than-normal molarity of Tris base in the running gel (0.75M rather than 0.5M)? The blob appears shortly after the samples enter the running gel. All DNA was removed prior to boiling. Neither of the gels had any yellow in them. Thanks again!