u/UnpaidNewscast

I know cottage cheese is a low point food (0 points if it's 0% fat), and it is generally low in calories, but I cannot have it in my home.

A few days ago I bought some thinking I could resist, and eat it only in moderation. That lasted through day 1 and day 2.

But yesterday, on day 3, late at night, I took a spoon to the container and ate everything to the very last curd. I still logged it and it took up half of my daily allotment points, when I already used all my points and some of my weeklies before this.

I'm not too beaten down by this, but I know for sure now: I cannot have cottage cheese in my home.

I am a PhD student (1st year, straight from undergrad) and I'm so fed up with my current failures. I have been trying to do the same western blot for a month and a half. I feel like I'm wasting the valuable time I have over the summer before classes start as well as wasting $$$ and disappointing my PI who is the nicest person ever.

I've troubleshooted almost every aspect of the western process. First, it was dodgy gels because of a batch issue. Once that got I figured that out after a week, it was transfer issues. Bubbles and incomplete transfers galore! I figured out what I was doing wrong and eventually got it right, but it was painful.

Finally I got to antibody incubation and: A billion non-specific bands! Nothing was distinguishable, so I reran it a few times, changing protein concentration and the amount of sample loaded since my PI said it was a potential issue. None of those changes made any difference. Then my PI said maybe its the antibody that I was using, and that I should try to see if my process works with others. So I did that: AND SAME THING! So its me, not the antibody.

I play around a little bit more; stripping membranes and the stripping protocol won't work, altering antibody dilutions to see if that will dim the non-specifics, change my incubation and wash times, etc. Nothing works.

Finally my PI checks my math for the protein concentration loaded per sample and...I did the math wrong. I felt so stupid but I knew it was an easy fix. Surely now, with new corrected protein concentrations and an appropriate amount loaded, everything would be smooth sailing, right?

I load an appropriate (for my protein of interest) 20 ug and wait. I'm careful in my transfer and on top of my washes and incubation. I time everything perfectly and when I finally go image: nonspecific bands.

"But it's okay" I think; the band I'm interested in is much brighter than the noise. Even though the vehicle control which is supposed to have the least amount is the brightest...and the one that should have the most, theoretically, is not there at all. Still, I am happy that the result is at least somewhat different from my other attempts. Since my protein of interest is the catalytically active form, I move to strip and see how it differs from the catalytically inactive form so I can get a rough idea of what's happening in my samples. Not a big shock that my strip fails in the first round, but it gets through the second.

I strip it and, because I am an unfocused person in general, don't realize that I swap the cut membrane that has my protein of interest with the cut membrane that has my housekeeping gene. I sadly incubate each membrane in the wrong antibody. Today when I go to image, I knew something looked wrong but I couldn't figure out what. I notice the notches on my membranes don't make any sense with what I assigned and my protein of interest was suddenly 20 kDa larger than expected.

I am so over this bad luck and my own mistakes for not paying attention, but I am a stubborn person. I just make 4 new gels. I load 4 gels. And in every gel, there is 2-3 wells broken. I wasn't able to tell if they were broken until after loading, and now I am sadly running gels that probably shouldn't even be transferred. And, no, I didn't poke the gels when loading (that's one of the few things I'm actually good at not doing!).

I just want to cry. Everything has been such a failure. I've been having actual nightmares when I sleep about non-specific bands and my PI calling me an idiot (even though, like I said, he is super nice and would never!). This has caused me so much anxiety to the point of somatic symptoms like headache, nausea, and vomiting. I want to avoid lab so I can avoid the failure, but at the same time I desperately want to master the western blot. I've never felt so dumb in my life.