This is a summary of diffusion experiments with A3G that were conducted during the week of 5/10/2010-5/14/2010. The HMM structure of A3G is a large protein multimer held together with RNA. Since the ultimate goal of this project is to develop a screening assay for HIV drugs, a successful experiment is one that will show…
These experiments were conducted during the week of 5/10/2010 – 5/14/2010. To recap on this project, Barrett and I have been working with Dr. Harold Smith in the hopes of developing a drug screening test that consists of small-scale diffusions. In these experiments, we are working with APOBEC3G (A3G), a protein-RNA complex that is active…
I recently began working directly with Dr. Harold Smith’s A3G protein. In my previous posts, I worked with BSA and IgG in order to develop a small-scale diffusion set-up able to reach equilibrium before it dries out. These preliminary steps were working towards my ultimate design for a quick and accurate diffusion set-up for A3G….
During the past few weeks, I have been experimenting with different array setups for diffusion. I have been working with BSA, but I will move to APOBEC3G protein once the arrays Barrett designed are finished. All of the pnc-Si arrays used were treated with UV-ozone. Treating with UV-ozone cleans the surface and helps prevents the…
In my last diffusion experiment, I used wafer SC 442. The results seemed to show that IgG was indeed diffusing through the membrane. As a follow up experiment, I am trying to show that I can pass IgG through more than just the one membrane. Furthermore, SC 505 has a much larger average pore size…
The results of my last diffusion experiment regarding BSA and filtered IgG (using SC 364) were inconclusive. (See “Diffusion Test– filtered IgG and BSA”) Looking at the histogram of results, there is no clear difference between the absorption of filtered IgG and buffer A. For this follow up experiment, I used SC 442. Of the…
The human IgG did not seem to diffuse through the membrane the first time we conducted a diffusion experiment. Barrett believed that the lack of diffusion was most likely due to protein aggregates blocking the pores. So, the human IgG was filtered, and this post is the result of a second round of diffusion experiments….
SepCon Experiment (Carrie): Does it work in a SepCon format? Use “Protein A Beads and IgG Binding and Filtering (with SepCon Format)” 9/29/09 Make Buffer B at pH 3 9/30/09 1) Rinse off ethanol 2) Add IgG Take absorbance reading of 1 aliquot of IgG (2×10μl samples) [D7= 0.565abs, D8=0.63abs] Transfer beads to 2ml centrifuge…
Test of Binding/ Unbinding (Carrie and Barrett): Test to see if we can successfully bind Human IgG to the protein A beads and then remove it with buffer B. After ordering the new human IgG and protein A beads, I have been working on a higher-quality analysis of the binding and dissociating of the IgG…
Last week, Barrett and I made up the three buffers needed to perform this test: Dissociating buffer: 0.1 M glycine-HCl, pH 3.5 1 M Tris Binding buffer: 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.5 In addition, I made a solution of protein A beads that consists of 0.0166 g of beads in 50…