Blocking Buffer Room Temperature Phase

On Friday (February 28th), I finally started my project for real! Today I ran the room temperature phase. At room temperature, Casein and BSA were each blocked for times of 30 minutes, 1 hour, and 2 hours- Casein at 1/2x and 1x and BSA at 0.2, 1, 2.5, and 5%. The slides I ran today are indicated in yellow on the schedule below. I also added a control slide that was blocked with PBS for 2 hours.

My protocol was as follows:

1) Add 1mL of the specified protein solution to the specified slide.
2) Block on the shaker for the specified time.
3) Drain well with pipette into waste container.
4) Wash slide with 4 mL PBS for 5 minutes.
5) Drain well with pipette into waste container.
6) Transfer slide to new plate.
7) Stain with 4 mL blue stain for 15 minutes.
8) Drain stain into waste.
9) Wash slide with 4 mL PBS for 5 minutes.
10) Air dry slides for 5 minutes on paper towel.
11) Write blocking solution, blocking time, and temperature on back of slides.

When staining, the blue dye clotted with 5% BSA blocked for 30 minutes, 5%, 2.5%, and 1% BSA blocked for 1 hour, and 1% BSA blocked for 2 hours. In the future, we will work to find the cause of this clotting.

In this image, the 1 and 2 hour blocking time slides are still blocking with their respective protein solutions (step 2). The 30 minutes blocking time slides are being stained with blue dye (step 7). You can see the clotting of the 5% BSA slide on the far right.

I also had a photographer in the lab today, which added to the excitement of the day! I can't wait to continue my experiment in the coming weeks and begin to analyze the results.