Maddie Yeo

9 Questions 11 Answers 0 Followers

Questions related from Maddie Yeo

What should I do to detect phospho proteins in western blot and prevent non-specific binding?

Hi all, I am trying to detect p-IRF3, p-STAT1 and p-STAT3 proteins. When I used 5% milk with TBST to block my membrane and dilute my primary and secondary anti-phospho antibodies, I got back a...

29 October 2021 7,058 2 View

Will protease and phosphatase inhibitors still work after it's expiry date?

Hi all, my lab currently uses thermofisher's Pierce protease and phosphatase inhibitor mini tablet. I noticed that the expiry date was back in May 2020. I keep the tablets at 4°C, and dissolve 1...

29 October 2021 1,490 3 View

Why can't I detect any phospho proteins on my western blot?

I repeated my western blot 5 times with 5 different samples, trying to detect phospho-IRF3, phospho-STAT1 and phospho-STAT3. Sadly, all 5 of them had totally no bands present for all 3 proteins...

21 October 2021 5,368 4 View

How long can I keep my western blot samples at 4°C for, after adding loading dye, DTT and boiling for 5mins?

I'm looking at phosphorylated proteins in triple negative breast cancer cells, so they are quite sensitive. I was wondering how long can I store my samples at 4°C after mixing them with loading...

19 October 2021 4,733 4 View

Will having foam/ bubbles during cell lysis affect western blot quality?

I spin down my cells with PBS, then discard the PBS and add RIPA lysis buffer to lyse them. However, I always have foam/ a lot of bubbles forming whenever I add the lysis buffer and resuspend my...

15 October 2021 3,997 3 View

How to make working concentrations of a drug if diluted in endotoxin free water?

The drug I am using needs to be diluted in endotoxin free water in order to get the working solutions. I am thinking of seeding my cells (in cell culture media) in 10cm plates the day before, and...

01 January 1970 7,930 4 View

How to detect phospho-IRF3 protein in MDA-MB-231 and/or BT-549 cell lines?

Hello everyone, I am trying to detect pIRF3 protein in MDA-MB-231 and BT-549 cells, but have been unsuccessful for a while now. I am able to detect other proteins such as pSTAT1 and pSTAT3, but...

01 January 1970 6,837 2 View

Will centrifuging my samples at RPM instead of g impact sample quality?

Hi all, I am using M-PER Mammalian Protein Extraction Lysis Reagent to lyse my cells for western blot. The protocol given was to spin the cells down at 14,000g for 15mins and collect the...

01 January 1970 9,955 8 View

What will happen if I harvested cells in media containing drug?

I treated my cells with Bafetinib for 2h, 4h and 8hrs. I harvested my cells at the specified timepoints by scrapping the adherent cells in the media containing Bafetinib, followed by 2 washes with...

01 January 1970 1,781 5 View