Jada Myricks- Chaiken Lab- Week 5 08/11/2014

Week 5 Back to the Drawing Board

Viral Gradient Centrifugation and p24 ELISA plates

Since our previous work wasn't as successful we are going to repeat the same processes in hopes that our results will be more predictable. We began the with the viral gradient centrifugation. I established a gradient using two liquids of different densities One was at 20% and the other at 5%. I began to fill the centrifuge tubes slowly with the 5% and then little by little added the 20% liquid. This produces a steady increase from 5% to 20% by adding a higher concentration in increments until it reaches the 20% concentration. After 12 tubes of these gradients were made I moved to pipeting the virus just on the surface of the liquid so that it wouldn't mix before centrifugation. I loaded all 12 of the tubes in the centrifuge and let them spin for 2 hrs.

After the centrifuge I took out the tubes and removed the first two layers of liquid. These first two levels would most likely not be useful because they would almost entirely just be virus that didn't distribute throughout the tube. Next I pipeted just below the surface of the solution and filled the wells on the plate making sure I kept track of which levels I added to each well. It was really important that I didn't pipet too far down in the tube because that would disrupt the gradient and then the tube could not be used.

Then these plated samples were put into the freezer overnight for the p24 ELISA plates I would be doing. It is best to have them sit overnight because you want as many of the viruses as possible to stick to the ELISA plates for a clearer reading.

The next day I did the usual ELISA process. I block all sites not occupied by virus with BSA. After that I added the primary antibody and that was placed on the shaker of 1 hr. After that I went off to lunch with Dr. Crider and Alex!!!!! Yum :)

Then I did 3 washes to rid the plate of any thing besides what was to be detected. Then I added the secondary antibody which is conjugated to an enzyme. This enzyme produces a colormetric detection when its substrate is added to the sample. I put the results in the plate reader and this is what I got! This is a much better reading than the one before, but there are some mysterious inconsistencies that need to be figured out.

In the rows with the green corners are the averages of each set and then by column the are grouped by kind. They should be similar from top to bottom and should dwindle from left to right excluding the last two columns which are controls. So while this assay shows a slight trend there must be something going on with the KR-13 molecule itself and not just the virus.

Thanks!
-Jada