In this gel I evaluated the linear range of coomassie brilliant blue. From this test it looks like things are linear from .0156 to .125mg/mL. My guess is that the protein ladder bands in the previous experiment fall within the range. I used the Bradford assay to find the total concentration of the stock protein…
Previously I have silver stained all gels because the separations look very clear with this staining technique. However, the silver stain is not giving very quantifiable results. Coomassie staining means a loss in sensitivity, but it’s the gold standard for quantifying protein amounts. Here I’ve stained one of the protein separation results and rerun with…
I used the transwell track etched membranes for these permeability experiments. I first looked at 5nm gold in 10mM KCl. After 24 hours the retentate and filtrate had just about equilibrated: We can compare these scans to the stock 5nm stock solution and see that the height of the 525nm peak is related to the…
I wanted to have 3 repeats of the previous salt-tuned separations. I’ve gone through and performed the densitometry on a couple more gels and compiled the results. 100mM: The first two experiments were from the two experiments set up on the same day. The third (yellow triangle) was set up a different day and does…
Separations were performed at 2 different salt concentrations with equal volume filtrate and retentate (to avoid dilution problem). Separation time was 40hrs rather than the usual 24. The lower salt concentration run has a lower cutoff – which is just like what we see in the DNA separations. Remember though that proteins have different charges…
UPDATED: More sizing characterization (see chart and figure below). We recently purchased our own gold ladder, and today I’ve done some of the initial characterization. Particles come in 5, 10, 15, 20, 30, and 40nm. Here are the absorbance scans in both water and 10mM KCl: The scans look similar, so none of these particles…
PES Membranes: I tested 100 and 300kD PES membranes from Pall. 5 and 20 nm particles were used in both water and 10mM KCl. I won’t show all of the data, but I didn’t see any particles in any of the filtrates by absorbance or DLS. The filters were also stained red after the experiments….
The new electroosmosis wafers have randomly distributed chips with 1, 3, 6, or 9 .1x.1mm freestanding pnc-Si squares. The idea is that we can use these to get a better EO rate vs. active area curve. SC125 seemed to have some sort of film on it, but I decided to give it a shot anyhow…
The BT method mentioned in this post seemed to give stable particles, but the BSA-Au did not work in separations. After about 24 hours, the particles retained pink color but seemed to settle in tubes. This may be due to reversable flocculation, not aggregation. The retentate and filtrates in separation experiments both lost all color,…
This was mentioned in the meeting today, and I’ll try to describe what’s going on here. We start out with a system with a smaller volume on the top of the membrane: If we start out with 10 mg/mL in the retentate, and assume perfect equilibrium of the protein/particle: The concentrations in each compartment would…