Happy belated Father's Day to everyone!
DAY 3 RE-CAP (remember that I actually started counting from Day 0)
I was in lab on Sunday (Day 3). I did not have to cell count or collect yesterday (only every 2 days), but I was in here to check on my cells and also to see the Beta-tubulin blot & complete the protein lysate from the day before (Sat, Day 2). I made more RIPA for the collections where I wasn't able to get all of the lysate easily (Days 30 & 70).
|Protein Lysate success! Finally!|
|Time to prep 27 uL RIPA + 3 uL Lysate--> quantification|
|Carefully pipette in 9uL in doubles of RIPA Standards & actual samples, then distribute 200uL of BCA assay into each well|
|Incubate your new baby for 30 mins!|
After finally getting the protein lysate for all remaining collections I want to look at (Day 30, 60 70, & 74), I was able to quantify the protein.
My colleague made a really good point about the bands on my last Western not showing up as distinctly as I would have liked because I'm using the Blotting Chemi ultra sensitivi setting when I visualize, and the reagents we use work best with Densitometry-->Photographs-->Invert. I used Densitometry for the Beta-tubulin blot and the bands do appear more distinctly, so, I threw my blot back into the 4 degree fridge to rock overnight again with some Primary Antibody (AR-N20) on it.
Called it a day and headed home!
Today is Day 4.
I don't have to split today, but I will have to split my plates on the next collection (Day 6). Splitting plates takes a long time, so I have to cushion out appropriate time to do everything in a given day.
So for Day 4, I took the cell photos in black and white at 4x, 10x, 20x, & 40x for both the FBS Day 4 & ADT Day 4 plates.
After counting and collecting them, I also revisualized my WB from Fri (Collection 3 D0-12).
This time, I washed for 15 mins (instead of 10). The background was a bit messy last time, so I washed 3 x for 15 mins each, threw on Secondary Antibody for 10 mins, and then washed 3x again for 15 mins each.
Photos and raw cell count of both Day 2 & 4 follow:
[EDIT JUNE 29TH, 2016 : TAKING OUT THE RESULTS FROM THIS BLOG UNTIL I HAVE MY THESIS ACCEPTED AND PUBLISHED. LAB DIARY WILL BE MOSTLY DOCUMENTING DAILY LABLIFE, PROTOCOLS, + TROUBLESHOOTING FROM HERE ON OUT.]
Comparing FBS to ADT side-by-side: