Hi! I'm working with cryopreserved tissue samples and am trying to double label them for reelin in the cytoplasm and another protein in the nucleus. I need to be able to distinguish individual cells when they're visualized by their reelin. Background staining makes this difficult, would using more Triton make the cells look fuzzier as the membrane would be more permeable? Or would it reduce background noise by making the surrounding tissue easier to clear and making the intracellular reelin easier for antibodies to bind + provide contrast to the background? I'm currently using 0.1% Triton. I can't figure out what would happen between these two options if I upped it to 0.5% for the antibody incubation.

Hi everyone,

I’m wondering if anyone has done a successful Co-IP in transiently transfected A7R5 cells?

My biggest issues are that even with 2 x 100mm dishes I don’t get a full 1mg of protein in the Lysate, plus getting really low transfection efficiency.

I use a DNA to JetPrime ratio of (1:3), transfecting 10μg per 100mm dish, let the cells transfect for 24 hours then refresh media and let the cells recover for an additional 24 hours, treating them with inhibitor 1 at 16 hours prior to lysis and inhibitor 2 at 4hrs prior to lysis.

Wash cells with cold DPBS

Use ~300-400ul CST 1x lysis buffer (+ 1x PI cocktail) on each dish for 15 min in 4C, Sonicate, and spin down at 4C. Quantify using Pierce BCA assay.

Preclear 500ug of lysate using blank A/G magnetic beads, then transfer Lysate to A/G magnetic beads plus the mouse Myc-tagged Ab (calculated based off the manufacturer 1:250 recommendation for IP). I then let the Lysate rotate overnight at 4C slowly.

Wash beads the next day with the same lysis buffer twice. Add 20ul of 4x Lammeli buffer and boil for 5 min. Run it through 8% SDS Page gel, transfer, block in 5% BSA, and probe overnight with rabbit Ab at 1:4000 (according to trial and error on normal WB and not IP). Then secondary ab 1:5000. When I image I get bands even in the non transfected lanes.

I’ve been struggling with this for a year and desperately need help.

Please any and all help is welcome!

…in a place where the PI have invested a lot of time with you?

& that the case is I feel tired of the pressure,
was told “take it easy” but not really,
and need some rest but not given?

Rao Yi at Peking university has accused on chinese social media that an editor (Steve Mao) at Cancer Cell is running bribing schemes, alleging that this editor has solicited publication for a fee of $2m RMB.

Original post in chinese linked

There’s a couple of bioinformatics tools that I read about in papers and no one in my lab is familiar with them, so I decided to self learning, I enrolled in a course in coursera but it is kinda above of my current knowledge. I studied computational biology grad course but my knowledge is not applicable in lab yet, since I didn’t do any computational work for my thesis. So I am currently creating my own learning plan and would appreciate any advice and/or tools or resources to help me. I specialise in xenobiotics metabolism but open to learning anything.

Hi :) I'm not sure if this is the right place for this discussion, so apologies if not! Also the title is not a pun I promise

Let me begin with the disclaimer that this rant is largely a gender issue, as I am a woman working in research.

I'm currently in the process of getting my masters, this is my second lab I've worked in (from molecular bio to behav neurosci) and I've noticed that despite being very different labs, they have similar issues; namely, that a lot of guys who come in do not respect the women they work with.

Of course, obligatory disclaimer, not all guys, I've worked with plenty of perfectly wonderful guys.

But, I've also had a reoccurring issue of men not taking me seriously, not listening to me, even when I'm in a position of authority (I was one of two senior students in my previous lab, and am now lab manager in my current lab).

Earlier last week I was giving a lecture on our study premise, plans for the future etc. and one guy literally had his headphones in the whole time, on his phone, etc. 😭 I've had to deal with guys like this so many times I literally have a "procedure" to deal with them that I call potty training, lol.

Is this just what it's like to be a girl in this field?? Will people actually respect me if I have a PhD?? Am I doing something wrong??

I really feel the need to emphasize I have worked with plenty of perfectly respectful guys who I loved to be around, it's just also my experience that so many men I've worked with have an ego, think they know everything, etc. I'm sorry to the guys reading this I'm sure you're great lmao

edit: thank you for the kind words/commiseration!! I'm very open to advice especially from other lab girls :)

Hi all! Dealing with a bit of compassion fatigue and every time I come on here and stumble into a positive comment on animal work it eases my feelings a bit. For example I saw someone say “I love working with the little mousies” and that made me happy. I call them mousies too. Or when I hear about the treats they get etc. It’s nice to know others care and are being kind to their animals.

Can't post video so I post the screen capture.

https://www.reddit.com/r/interesting/comments/1x94jxc/two_brain_cells_connecting_to_make_a_new_pathway/

Hope I could make some of you giggle hehe

I've recently graduated from Biomedicine, and before getting into a master I'd like to work in a lab group for some time, even if it's just preparing samples. I'd like to try to do so in a foreign european country (I'm from Spain) as in Spain there are little opportunities. Has anyone done this? Started working as a lab tech after graduating from collage in a foreign country (or something remotely similar)? If so, I'd very much appreciate some help on how to start😅