We have recently been discussing the best way to analyze the diffusion and SepCon separation gels. In the following figures, I present line scans of the gels. These appear to be a bit noisy and are somewhat misaligned due to variations along the length of the gel. Blue = Control, Green = Retentate, Red =…
Using wafer 302 I set up 2 20nm and 2 50nm gold diffusion experiments. After 24 hrs I removed the retentate and filtrate and looked for the gold using the Malvern Zetasizer. We can’t perform the diffusion with both 20nm and 50nm gold because the Zetasizer cannot differentiate between the two sizes. r value Sample…
My previous post outlined the results of a EQ format separations using membranes from wafer 319. I have followed up that experiment using the same membranes, but in a diffusion based separation. I separated 2uL of 9.3 mg/mL clarified brain extract using a reservoir of 60uL. I allowed the diffusion process to occur overnight, and…
We finally achieved a separation using the EQ format. 4 SepCons from wafer 319 were loaded with 500 uL of .93 mg/mL clarified brain extract. They were run at 1000 rpm (which we roughly figured to be 3.3 psi initially) for 2 hours. After 2 hours, I judged the flow through the membrane to be…
Previously I had posted a chart depicting the permeabilities of SepCon membranes from w310. I went back and noticed some errors in the equations. Here is an updated version of that chart. Also included is a sample where I used the ultracentrifuge to clarify the protein right before the experiment, which should remove any larger…
Last week I performed spins with SepCons from w310. I tracked permeability and tried to perform some separations with proteins. w310 had high air permeability (link), but from my diffusion data it looked looked like the cutoff was possibly between beta-galactosidase and phosphorylase b (link). Myosin definitely did not make it through these membranes, but…
This is some really preliminary data that I hope to add to in the next couple of days. Yesterday I prepared 4 SepCon samples from wafer 310. I spun them at 3.3 psi for 2 hours with just PBS. I recorded the permeability, and then performed the exact same test with 3mg/mL protein in the…
This week I set up some diffusion experiments with the inner membranes of wafers 187 and 310. From the TEM images posted previously, we see that these inner membranes have what appear to be either very small pores or small divots that do not pass entirely through the membrane. I allowed the protein standards to…
Link to “A closer look at separations” We recently bought purified proteins that I will use to build a molecular weight ladder for membrane separations. I initially dissolved these proteins in salt solution and spun in an ultracentrifuge to remove aggregations. I then sized the proteins using our Malvern Zetasizer, which gives hydrodynamic radius, assuming…
We have previously discussed how the membranes near the center of the wafer have very small pores, while as you progress outward larger pores begin to appear. I tested one of these central membranes from wafer 304 (TEM sample, position -1,0) using our standard fluorescence microscope based assay. I observed obvious rhodamine diffusion through this…