Hello,
Please see attached a snippet of my western blot for this inquiry. Some brief background on this particular sample ("1"): This sample is a Mycobacterium spp. cell culture that was heat-killed in a CL3 lab prior to me obtaining it. I performed total protein extraction on the heat-killed culture using a urea-based lysis buffer + TBP + protease inhibitor and sonicated with a probe (10 min total (5 min sonication) at 100% amplitude, alternating 1 min on, 1 min off) followed by sonication in a cup-horn (1 hour total (30 min sonication) at 100% amplitude, alternating 5 min on, 5 min off). I then "cleaned" the sample up using a commercial clean-up kit and loaded 15 ug of the sample into lanes of my SDS-PAGE gel to test different primary sera and secondary antibody concentrations to optimize the western. As you can see, everywhere this particular sample is loaded (wells designated with a red, bold "1"), I get a smear from top to bottom throughout the entire lane. Does anybody know why this is? I am ideally expecting banding from the various sized bacterial proteins but clearly this is not the case. Are the proteins being degraded from the heat inactivation prior to me obtaining the cells? After heat inactivation, they are kept in the incubator for a few weeks to confirm sterility of the cells before they leave CL3. Mycobacteria grow very slowly so they could be in the incubator without protease inhibitors for many weeks. Is the lysis of the cells incomplete? Mycobacteria also have hardy cell walls so maybe my lysis protocol is not sufficient? Any ideas would be helpful. Thank you in advance!
~ Cindy
I have a few technical questions:
1. How did you clean the samples? Are you using a resin to bind the protein or just to remove cell debris?
2. How much urea is in your lysis buffer? Are you removing most, if not all, of the urea prior to loading on the gel? Does your "clean-up" kit remove the the urea? If the urea concentration is high, I suggest you dialyze protein (using the small column type of dialyzers where you can load 0.5mL) or precipitate the protein to remove the urea.
3. Have you tried loading different protein concentrations on your gel? Based on your gel, the protein looked overloaded. Try loading 1-2ug and see if you obtain the same result.
4. Your other samples do not look too great neither. I see smearing in thos lanes too. Are you hand casting your gels or purchasing commercial gels? To help improve your protein migration, check to make sure your gel pouring or pre-cast gels did not expire.
As for protein degrading during your lysis prep, I am not too sure. I don't think your protein is being degraded because you have protease inhibitor in the lysis buffer.
I think your issue is mostly technical either due to high urea, gel issue, or overloaded protein. However to check if your protein is being degraded during the prep via heat or mechanical degradation, spike-in any protein with a tag that you can detect via western, such as FLAG or His. Run your spiked-in sample through the normal protocol, isolate protein and detect the spike-in protein. If the protein looks ok and not degraded, then your issue is one or more of the listed ones above.
Dear Cindy,
To me, it seems that your samples have any contamination leading to smear, it could be either metall ions or polysaccharides, as well the Urea as suggested above. Try to precipitate your probes with TCA prior electrophoresis.
This is not the degradation of proteins.
Dear Cindy,
I also work with mycobacteria for my project, but I use M. smegmatis instead. I am amazed you use over 1 h sonication to lyse your bacteria. Even though mycobacteria possess a thick cell envelope, from my experience, usually 2 to 5 min. sonication should be enough to achieve full lysis of the bacteria. Avoiding oversonication is very important, specially if your target protein is membrane-bound. MPs are prone to thermal-dependent aggregation, even after being exposed to harsh detergents, such as SDS.
Smearing can also occur if you:
1) overload the gel with too much protein
2) Your protein sample contains rests of cell wall, lipids or genomic DNA which affect protein migration. This can be solved as recommended by Airat with protein precipitation with TCA or after Wessel-Flügge, i.e. Methanol-Chloroform precipitation
3) If your lysis buffer contains other detergents than SDS, the ratio of detergent to SDS might severly impact the migration of your protein.
I also link this paper which might help
Article Common Artifacts and Mistakes Made in Electrophoresis
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