Pressurized Movable Cutoffs
While wafers 670 and 673 did not hold up to diffusion experiments, they were strong enough to perform pressurized separations using the pressure cell. Each experiment lasted for 40min, although the average flow was only about 5ul for this duration.
673 has a pore cutoff around 30nm (refer to Dave’s post). The following gel shows albumin as the last protein to significantly pass into the filtrate – meaning the cutoff is above 67kD. Previous membranes have had much higher cutoffs than these in diffusion – myosin used to be the only retained protein (200kD on the gel).
W670 has a lower pore cutoff around 20nm. The flow rate was lower for this membrane, only about 1-2 ul in 40min, and it appears that the second trial didn’t work (there was some evaporation or loss of the wetting fluid by the end of the experiment). We see in the first trial though that the albumin band is not as dark at in 673, meaning that thee cutoff is potentially somewhere in between ovalbumin (45kd) and bsa (67kd).
*We may need to discuss more what these cutoffs mean if the larger proteins are in fact in complexes in these mixtures.*
I’m convinced that the cut-off is lower than it has been in a long time. But I’m not convinced that w670 and w673 have different cut-offs. More experiments would help …
The second gel is not really the quality I like, but it seems that there may still be a shift in the cutoff here.
Chris mentioned that there were other wafers that might have even lower pore cutoffs but haven’t been TEMed yet. Can we get our hands on these?
Why is the flow so low? Were these ozone treated? Are the pure water flow rates and air flow rates posted somewhere? I thought these were reasonably porous, so this is surprising. At these volumes, diffusion may be the dominant transport mechanism…
The gels and cutoffs do look nice.
You’re right, we may just be seeing diffusion in these experiments because of the low flow rates.
We haven’t done flow rates with these membranes because of the limited samples. If they follow the trend though, I’d expect ~70 ul to pass in 40 min. However we know that PBS slows things down, and so does a concentrated protein solution. I am not surprised by these flow rates.