Blocking Buffer Results!

Last Friday (April 25th), I finally analyzed my results from the room temperature and 37C runs of my blocking buffer experiment!! First, I took pictures of all of the slides in groups of same blocking temperature and same blocking time. To quantify the results of this experiment, the slides were scanned for fluorescence with a 488nm laser. This imaging resulted in the images below, in which the shading of the slides are actually inverted, i.e. the darkest slide in this image is actually the lightest slide.

Scanner image for room temperature slides

Scanner image for 37C Slides 

The resulting images were then analyzed for mean intensity of each slide using a program called ImageJ. This data was then adjusted to account for the inversion of the images and the background noise of the control slide. The following images show graphical representations of the mean intensity/slide area, where intensity indicates lack of dye, or the amount of protein bound to the slide.

From this data, the optimal blocking conditions for BSA are 2.% for 1 hour at room temperature. There is a general trend for BSA that the amount of protein bound for a given temperature and time period is maximized at 2.5% BSA and decreases for 5% BSA. The only exception to that trend is this trial was the 37C 1 hour data, which may be due to splotching on the slide affecting the mean intensity measurement. The optimal blocking conditions for casein are 1x for 2 hours at room temperature. The amount of casein bound increases with concentration at all time periods at room temperature. Casein does not bind to the slide at 37C.

I can't wait to present my work to the Emma community at the student achievement assembly on April 30th!