Attached is the picture of the gel in question. In Lane #2 for example, there appears to be multiple dye fronts forming. Lanes 9 and 10 are what it is supposed to look like and it usually does. The issue is that when the smears form and the dye front seems like form a "hill" like pattern in lanes 4 and 5 the corresponding western blot is completely uninterruptible as the proteins don't stack in a band at all.
Has anyone seen this before and been able to identify the cause?
I checked the running buffer and its the usual pH 8.3. The tank is full as well. We are using precast 4-20% gels from BioRad and haven't had any issue with them before.
The only thing I can say is that the protein concentration is quite high but I am only loading ~30 ug based on my BCA assay which I have done without issue for months. Could this be a result of DNA contamination or protein aggregation possibly. I did try centrifuging the samples at 13,000 RPM for 15 mins before loading and it did NOT prevent this from happening.
I boiled the samples at ~95 deg for 10 minutes in 1x laemmli buffer with 10% BME as per the BioRad guidelines.
Check whether your sample contains high salt concentrations or components that may change the pH. It also looks like your chamber is leaking and the level of running buffer is dropping below the rim of the gel.
Also rinse the wells a few times with your running buffer prior to loading anything. There tends to be stuff in there that disturbs the run.
Did you see precipitations after the centrifugation? After the denaturation?
Do you load whole cell lysates here?
Thanks for the helpful suggestions. The picture of this gel was not taken with it in the tank in running buffer. When the problems occurred, I stopped the run and took a picture of the gel on a light table which is why those yellow bands appear. There were not bubbles in the wells during the run and I did thoroughly rinse out the wells.
I had some IP samples on another gel at the same time and they stacked fine leading me to believe it is 100% an issue with the samples here which are whole cell lysates. The cell debris was cleared with centrifugation but maybe some remained. Perhaps too high a protein concentration and/or too high salt because of the high concentration of the lysate. For the future I will lyse the same sized pellet of cells in 2-3x more cell lysis buffer volume. Adrian Marc Hauri Engelbert Buxbaum
Sounds good. What kind of lysis buffer do you usually use?
I myself once had the problem that my protein aggregated after the denaturation, as I boiled it too long and also the concentration of the sample was very high.
The bands were then smeared as you described it.
I remember another case, where I worked with a bacterial protein that formed stable trimers and it just did not run properly on my self-made SDS-gels. I then changed the gels and the running buffer and then it worked, but in your case that's probably not the problem.
I do not boil my proteins in sample buffer, but only heat them to 50 °C for 15 min. It seems to work better.
Check the pH of buffer as well as check the voltage . Load less amount of sample in well
I also met this problem and I am still working on it. The samples just do not compact to a line. Have you solved this problem?
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