A group of my classmates and I are looking into
Bifidobacterium longum, specifically the proteins present in the membrane and those secreted.
I am running cell membrane samples (post freeze-thaw lysis) as well as whole cells. Following the advice of my instructors, I did not add SDC as a lysis reagent and incubate prior to loading the samples. Instead, my instructor assured me the LDS in the 4x laemmli buffer i used would be a sufficient surfactant for the procedure (formulation on that product has it at 4.4 % LDS, and I loaded it in a 3/1 ratio sample to buffer, and samples were then heated for approximately 10 minutes at 95 celsius.
I am using a mini-protean tetra cell, and my ladder shows through just fine... I used a bradford assay to try to ensure protein concentrations were sufficient for the run, so the COMPLETE lack of results in all lanes loaded with samples makes me think the lysis process was not successful. Am I missing a step?
Keep in mind these samples are run through the premade stain free gels, and when this procedure is refined they will be digested in the gel, extracted, and sent to run through an LC-MS.
Thank you for your help!
Are you able to share with us a picture of the gel? We can help you more when we can see a visual.
Hi,
When trying to run membrane proteins on a PAGE do not boil (or heat) the samples before loading them. Boiling membrane protein samples in loading buffer tends to make them aggregate like crazy, and they will then get stuck in the well, note even entering the gel..
I don't know why you do not see anything with the whole cell samples, but boiling the samples definitely does not help with the membrane fractions.
I am currently trying another run, with a few variables changed (keep in mind these are just practice to nail down a solid method).
I will post a picture of the gel post run.
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