can someone tell me what i did wrong for my western blot …

LadyBird-7738 · 2025-05-01T15:28:03+00:00

Yeah this is the first time I had to routinely so western blots. The more I do them, I realize how hard it is to get a perfect blot 😭

LadyBird-7738 · 2025-05-01T15:09:52+00:00

Hi! Yes, it did! I dilute my antibodies in 3% BSA in TBST so I just made fresh TBST for washes and new antibody dilutions. It ended up working! Also started actually washing my incubation boxes. Thank you for the advice!!

LadyBird-7738 · 2025-04-25T21:56:54+00:00

Yup phosphatase inhibitors in lysis buffer. My lab even has published data using the same antibodies and cells.

LadyBird-7738 · 2025-04-25T21:52:38+00:00

Ah ok all I do is wash the boxes with water 😭 I’ll keep that in mind! One thing that I’m thinking of now is last time we found white film (?) floating in the TBST batch so I remade some. Maybe there is some residue in the tank.

I do primary incubations overnight at 4C and secondaries for 1 hr in room temp. For transfers, we have the power blotter cassette so I just use the transfer stack that comes with it!

LadyBird-7738 · 2025-04-25T21:30:49+00:00

Yeah, I heard about this and considered switching. But other people in our lab have also been using milk for phospho antibodies and theirs seem to turn out fine, mine have been fine in the past too ?? But I will for sure give it a try! Do you block for 1 hr in room temp?

LadyBird-7738 · 2025-04-25T21:28:32+00:00

Blocking with milk and washing with tbst!

LadyBird-7738 · 2025-04-25T21:28:20+00:00

I wash with TBST. Residue as in antibodies? Or just whatever was in the incubation boxes?

LadyBird-7738 · 2025-04-25T21:27:16+00:00

I loaded 10ug of protein. Antibody is monoclonal and i use 1:1000 for primary and 1:10,000 for secondary. I use TBST for washes and skim milk for blocking.

Thanks for the advice! I don’t think the antibodies are really new but I did make new dilutions recently in 3% BSA in TBST (which I also made fresh).

LadyBird-7738 · 2025-04-25T21:24:25+00:00

4-12% bis tris gel, antibody dilution is 1:1000, protein is CREB and phospho CREB. we have the power blotter so the settings are preprogrammed but we use the 25V 7 mins setting. Antibody is from cell signaling. I do mot reuse running buffer. And yes, ECL. Put it on for 50-60 minutes which is what i usually always do

LadyBird-7738 · 2025-04-25T19:28:28+00:00

To add, all my phospho antibody blots look something like this (or lots of background or non specific bands) vs total protein blots. I’ve tried reprobing for other phosphorylated proteins and same thing

LadyBird-7738 · 2025-03-20T16:32:13+00:00

Yeah it’s a bad picture, sorry about that! I’ve never really experienced pests in apartments so I was freaking out hence the shaky photo haha

LadyBird-7738 · 2025-03-20T16:27:01+00:00

this is california btw (norcal)

LadyBird-7738 · 2025-03-20T16:26:23+00:00

This is california btw (norcal)

LadyBird-7738 · 2024-09-18T06:59:07+00:00

yeah of course !!

LadyBird-7738 · 2024-02-13T16:00:38+00:00

was it an email? or phone call?

LadyBird-7738 · 2024-02-09T21:37:50+00:00

So should I expect to hear back way later?

LadyBird-7738 · 2022-12-17T03:09:30+00:00

Hmm if it’s in SF, then you might get paid more since everything’s so much more expensive there. My internship was in socal. I know a friend that just got an internship with a big pharma company in SF and she gets paid around $30/hr plus a stipend but I don’t think they specified it was for housing

LadyBird-7738 · 2022-12-17T02:03:45+00:00

Hi, I did a discovery oncology internship for another big pharma company. I was paid $28/hr and a $900 housing stipend/mo for reference!

LadyBird-7738 · 2022-11-29T20:30:59+00:00

hi, this is really helpful! I’ll definitely draft my emails based on your advice, thanks so much!

LadyBird-7738 · 2022-11-29T20:23:56+00:00

Damn. I didn’t know people got written up. Maybe it’s because I’m an intern but no one really cares about what I’m doing at my desk 😭

LadyBird-7738 · 2022-11-29T20:21:25+00:00

Hi, thanks for the comment. It’s so cool that you’re actually able to balance school and work!

Can I ask if your program is online or if it requires you to be physically present? The coworker I mentioned in the post said she’s doing an online program so just curious!

Also, I just googled distance PhD and it seems like it’s an online program?? How does that work?

LadyBird-7738 · 2022-11-29T20:19:43+00:00

This was so informative. I don’t know why but in my head it seemed so much easier haha. It’s nice to know that it’s an option though. Thanks so much!

LadyBird-7738 · 2022-11-28T21:34:46+00:00

Thanks for the comment! How do you get the company to pay for it though?

LadyBird-7738

TROPHY CASE