Additional Diffusion Assays
Cellulose Membranes
I set up a 24 hour diffusion experiment with Millipore 30kD cellulose membranes as shown in this picture:
After 24 hours I collected retentate and filtrate and visualized on the following gel:
I didn’t expect to see a separation so similar to our pnc-Si results in this format considering the extremely poor dialysis membrane experiments I did a very long time ago. However, the separation does not match the manufacturer’s cutoff. The Millipore membrane shows the reduction of species in the retentate that we haven’t really been seeing in our membrane lately.
Denature Proteins:
I attempted to perform a diffusion study of denatured proteins using the remaining RTP’ed 670 and 673 membranes. The proteins were denatured by combining 3 parts protein standards with 1 part 10% SDS/ 5% BME. I didn’t heat the proteins because the last time I tried that I ended up with fried eggs. Here are the results:
There doesn’t appear to be much of anything going through 670. Either something went wrong with this particular experiment (as I was unable to do this in duplicate) or the denatured proteins clogged the pores. It should be noted that 670 had a lower porosity than 673. All of the denatured proteins passed through 673. I verified the membrane to be intact, and the amount of protein retention supports this. It seems that in this case, denaturing allows the proteins to wiggle their way through, no matter the length.