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!
Can we see a picture. I am wondering if the 2 colours are actually one diffuse blob and the front part is meeting more acidic buffer near the bottom of the gel and changing colour because most of the common dyes can act as pH indicators turning yellow as the medium becomes more acidic which does happen during electrophoresis as the buffer is depleted
Hi, I think that you have some type of aggregation in your samples, for example if you have DNA the samples could be viscous and create a sort of blob. Did you sonicate the whole-cell lysates using in RIPA buffer? Did the blob appear immediately in stacking buffer or after? With 30ul of volume (even if you load only 20ug) I prefer to keep low the running voltage in stacking gel
Hi, thanks for the images. If I understand your questions you point out two possible problems.
Final Tris-HCl molarity in separating gel recipe (pH 8,8): you said that your final molarity (I assume you have a Tris-HCl stock solution of 1-1,5M) is 0,75M instead of 0,5M. Am I correct? If the answer is yes, from my experience I used a final molarity of 0,375 in my running gel, using the old Maniatis recipe, or the “lower-upper solution method”. I don’t have a degree in chemistry (I am terrible in chemistry!) but if you alter in some way the pH you can have problem as Dr. Rutland point out. If you use bromophenol blue (I premused) in your laemmli recipe maybe you see “some purple-blue-violet-indaco colour” for this reason (not yellow because it is not so acid); it is only an assumption.
For the RIPA buffer I don’t know your recipe as for laemmli one, where you used Tris-HCl at ph6,8. Check the molarity.
Even if you performed transfer step you can check with coomassie if the gel had some problem during the running step; if you attempt to analyze low molecular weight proteins your transfer time it was not too long.
Hi Allyson,
I have a couple of points for you.
- You are loading 30ul from a 20mg/ml protein extract, hence you are loading ~600ug in each lane. That is a very high quantity of protein, and I suspect that you are overloading your lanes! Typically people load 20-50ug of lysate/lane. This could be the cause of the blob that you are seeing, which is the condensation/stacking of the large amount of small proteins and peptides running just after the glycine that is running in the front in the separating gel. As you wrote above, you just started using RIPA; I don't know what you used before, but it is possible that using the previous method the efficiency of lysis was low, and therefore you were loading less material in each lane, and did not observe the blob.
- You specified that the MW of the proteins that you trying to separate is small, and you have been using 12% SDS-PAGE, which is not ideal for them (ideal to separate 25-150KDa proteins) . To enable you to focus on the smaller range, and to avoid from losing the small proteins, and get a better separation you have a few approaches:
Please see original entry for new update (12/8/17) pertaining to this image...
Hi, if we can exclude problem regarding the apparatus (cleaning etc.) and gel composition (Tris molarity, pH, etc.), I can only think about RIPA and laemmli buffers problem.
Can I ask you the recipe of the buffers that you load on this two last gels, also the one without picture? You write “1% Triton-X 0.25M 7.2pH Tris-glycine as a control against the RIPA” and I don’t understand. What do you mean for “2 separate batches” of 5x sample buffer? Do you froze the laemmli aliquots, when do you BME or DTT?
We have to exclude if some powders used is too old (SDS, bromophenol), if bromophenol concentration is ok, that the SDS concentration is not too high (5x laemmli and 30ul volume), sometimes if you add enough SDS in your lysis buffer you can avoid to add it in laemmli (I don’t like laemmli concentration above 4x, it is little tricky to make).
It is a little difficult to understand what is the problem because we don’t have a coomassie staining. The fact is, the blue cloud/blob interfere in some way with the proteins running or not? You have a diffusion, around dye front, caused by different variables, but do your proteins run well ? Do you have only to stop the gel before losing your small proteins or, instead, the running is a mess? Even if the WB images didn’t seem too bad.
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