I recently expressed my proteins with P. pastoris GS115 and then harvesting 1 mL samples for different induction times to see my effector proteins expression. So the protein samples are frozen at -20 in BMMY. I did an SDS PAGE with Laemmli buffer but the bands keep smearing like its very blurry. I already incubated at 95C for 6 minutes. I can see that the color is more black at longer induction time (always) but its always blurry. I did a side by side comparison to apoplastic fluid samples and they turned out fine.
I used TGS buffer and precast mini protean gel from BioRad. Is there something I need to do before the SDS PAGE?
Running the supernatant from Pichia in an SDS/PAGE gel will cause the lanes to run funny and potentially look blurry due to the components in BMMY.
How much of the supernatant did you load? Also, what percent SDS/PAGE gel did you run?
One thing you can do before running the gel (and this would be a big pain since you are screening time points to look for the expression of your protein) is to desalt or dialyze the sample to get rid of the media components. Of course you would need to use a membrane with a MWCO that would retain your protein.
Thanks for your answer. For each I loaded 15 uL samples with 1x laemmli buffer. What do you think of doing precipitation?
Ok 15ul is certainly not an excessive amount to load.
I am certainly not an expert when it comes to manipulating proteins but I do have a lot of experience with Pichia and expressing proteins. If you can do the precipitation, without it being a too much of hassle and you are comfortable doing it, then I say go for it. So long as you get rid of the components of the media (i.e. peptides, salts, etc.) then it would probably help with the gel running issues.
Ah, thanks so much! Yes since its important for me to see which induction time I think I will do the precipitation.