This is the protocol I use to measure band intensities. At the end of the explanation I will post and review my recent results concerning silver stain densitometry. First load a digitized gel into ImageJ Select Image>Type>8 bit to make it black and white. Select Invert to make the bands have positive intensities. Go to…
The following changes were made in this adsorption study: The MicroCons/NanoSeps were not spun. We will just characterize the differences between the materials. The pnc-Si membranes were assembled into SepCons because of the potential adsorption to the plastic surfaces. Mass standards were run on the gel in order to allow the measurement of protein concentration….
Previously I have shown that sample buffer can be used to lift adsorbed proteins off polymer membranes, which can then be imaged on a gel. I have used a similar procedure to assay adsorption on pnc-Si membranes. I used 4 samples from wafer 216 that all had pinholes. I incubated a drop of 1mg/mL BSA…
In anticipation of the pnc-Si separation paper, I have performed a couple of preliminary separations using NanoSeps and MicroCons. I applied 200ul of 1:20 brain extract ( ~.9mg/mL) to 100k NanoSeps and MicroCons, and I spun the samples for 3 minutes at 14000g. I had wanted to collect the retentate as is from the top,…
Previously we have studied protein adsorption to pnc-Si membranes by TEM and fluorescence. Neither of these methods will work with polymer membranes. Polymer membranes are too thick to view using TEM. Polymer membranes also autofluoresce creating a lot of noise while using our fluorescence method. Here I outline a new method of studying adsorption to…
It took me a little while to figure out how to turn the .avi file into a .gif, but here is an image of the diffusion through 501. Due to a problem with MatLab, it took about 5min before I started taking images of the diffusion. So this starts at ~5 min after the 100mM…
I recently set up 24 hour diffusion separations with the BioRad protein standards. The following gel shows the separations using w415 position (-4,3) and w416 (-1,3). Different cutoffs were observed with the two membranes.
We’ve recently put together our own protein ladder, which contains myosin heavy chain, beta-galactosidase, phosphorylase b, IgG, albumin, and cytochrome c. This is my first attempt running this ladder on w398, one of the new pinhole free membranes. From the TEM pics, it seems that w398 has pores all around the 50nm range, which is…
Most recently I’ve been working with colloidal gold nanoparticles (referred to as AuNP from here on). We chose to use AuNP for separations because it is an ideal hard sphere particle, unlike a floppy protein. We thought we could achieve more information about the cutoffs or the sieving characteristics of the membrane by using such…
UPDATE:Chris took the membrane sample and normalized with by the no spanning membrane sample. This was plotted against the theoretical spectrum of single crystal silicon. The curves have a similar shape, but the single crystal silicon has lower transmission. This may be due to the fact that it’s a perfect material or that there’s error…