SC 073, SC 074 Diffusion Separations

Two of the newer wafers, SC 073 and SC 074, came back with ~30 intact samples.  This would be more than enough for the separation work that I’m trying to finish, so I decided to see if they were any good.

SC 073 had an area in quadrant I that was caught in the middle of crystallization.

The inner samples though looked fully crystallized:

I used an inner sample from SC 073 for the separation.  Dave did RTP it at 800C for 5min to prevent ablation, but I was hoping since it looked fully crystallized this wouldn’t change the morphology much.

I’m not sure what experiment SC 074 was a result of, but it has a very low density of small pores.  I thought this would be interesting if we could achieve a low cutoff similar to the old blood protein separations.

Here are the results of my diffusion separations with protein standards. The image, the pore processing histogram, and the gel from the separation are shown.

SC 073 has a separation in between the 116 and 97 bands, which is reasonable given its pore distribution.  None of the protein on this gel made it over to the filtrate in SC 074.

One thing I’m going to try is to redo the SC 074 gel at a higher concentration of poly acrilimide to see if we can visualize any proteins down in the 10-20 kD range making it across.  This is where b2microglobulin resides, so there’s still the potential that this might work.

Small size resolution of SC 074

I ran the samples from the SC 074 on a 12% gel to see if there was a separation below the limit of the previous gel.  Here is the result:

In both trials you can see three light bands.  These correspond to 31 kD (carbonic anhydrase), 14 kD (lysozyme), and an unknown contaminant band in the standards.  We are probably not seeing 21 kD (tripsin inhibitor) because it is so light, but I’m not sure why 6.5 kD (aprotinin) doesn’t show up yet.  Maybe it’s also too light originally or it’s complexing.

Anyway, this shows that we are indeed seeing a separation with these little pores.  However not much is getting through in 24 hours.  Maybe the next thing to try would be to up the time of the separation to take a look at the kinetics.