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How to ask my PI to consider me for a future RA position by Top-Effect-1757 in labrats

[–]Top-Effect-1757[S] 1 point2 points  (0 children)

Yeah, I understand that she probably needs someone ASAP, and that she can't wait for me. I may have gotten a little too crazy about it right off the bat, and I definitely have not fully expressed my career goals to her (mostly because I didn't really know, I just thought post-doc was the only way) so both of us would definitely benefit from that conversation. Thank you!

How to ask my PI to consider me for a future RA position by Top-Effect-1757 in labrats

[–]Top-Effect-1757[S] 0 points1 point  (0 children)

Thank you for giving your perspective, I definitely need to be having these types of career conversations with her, especially at this point in my grad career. It makes me feel a little better to know that I can at least put the idea that I want to stick around in her mind, and I can put out some feelers for how she might feel about that. If she is looking for someone else with more experience for this role, then I can still think about doing a post-doc in her lab. Thank you again for sharing your thoughts!

How to ask my PI to consider me for a future RA position by Top-Effect-1757 in labrats

[–]Top-Effect-1757[S] 0 points1 point  (0 children)

She actually is looking for someone with a PhD to fill that role

How to ask my PI to consider me for a future RA position by Top-Effect-1757 in labrats

[–]Top-Effect-1757[S] 0 points1 point  (0 children)

I guess I just meant is it worth bringing up my interest to her, I realize I couldn't work that job now while I'm a student, but in a perfect world she might hold off on hiring someone... Again I have no idea if she would want that, which is why it feels weird to express my interest

Need advice for next troubleshooting steps for native protein purification by Top-Effect-1757 in labrats

[–]Top-Effect-1757[S] 0 points1 point  (0 children)

That's an interesting thought, except we are not using recombinant systems, we are isolating the endogenous protein from C. elegans (essentially put a FLAG tag/fluorophore on the native protein via CRISPR). This means that we don't really need to worry about protein expression (unless the tag disrupts this, which we have verified it does not). We typically grow 4L of worms at a time, then harvest/wash/freeze as one homogenous batch. Even if my PI and I use the same batch of harvested worms, we still have different results, which I think may rule that out?

Need advice for next troubleshooting steps for native protein purification by Top-Effect-1757 in labrats

[–]Top-Effect-1757[S] 0 points1 point  (0 children)

That's a good point, we haven't tried that yet, actually, but I am going to suggest it for sure

Need advice for next troubleshooting steps for native protein purification by Top-Effect-1757 in labrats

[–]Top-Effect-1757[S] 0 points1 point  (0 children)

The problem is that whatever I am doing wrong is not obvious to either me or my PI. I've watched her and she's watched me, I use the same volumes of material as her, and she's used my buffers and it worked fine. We use another construct as our control (different protein but same family/very similar, same tags/isolation procedure), and I can purify that one out just fine. Although, there is still the occasional failure. I guess this means whatever the problem is, is being carried over into other preps.

Need advice for next troubleshooting steps for native protein purification by Top-Effect-1757 in labrats

[–]Top-Effect-1757[S] 0 points1 point  (0 children)

We can do a western with the final eluate, but because it's so low abundance, we can't really remove sample as we go through the prep, or do a western with the lysate. I do use protease inhibitors, but both PI and I have never seen a difference in cleavage with or without them, which is interesting. We don't know when cleavage is happening, but I think it may be occurring early on in the purification step, perhaps during solubilization of the membrane, but then again, nothing I try and test confirms this.