video (this version is saved to the server. It’s smaller and blurrier) http://www.youtube.com/watch?v=2JZ8uTWhejs&feature=youtu.be (this version is private – it can only be accessed by people with the exact link – it’s high quality) I modified the bottom basket of a Sepcon by drilling a small hole into it. A Sepcon chip with an average pore size…
What I had in my two previous posts described as permeability graphs were in fact total volume passed/ total time passed graphs: What I thought I saw was that cake layer formation was dependent on the filter geometry. There is some justification for such a dependence in the literature – These folks found that spacer…
Using methods pretty much identical to those outlined in Tuesday’s post, I was able to get very similar data for a different BSA concentration. Whereas Tuesday’s curves were all done with 1mg/mL BSA, this was done with 0.5mg/mL BSA. The first time I collected sample from the 5 slot chip during the buffer separations, I let…
The following are TECAN spectrum of the filtrates (and a spectrum of unconjugated peptide) for a series of sequential separations performed on the Barcikowski peptide (CE12W) using 10, 20, and 30nm cutoff Sepcons performed last weekend. Note that all of this data was taken with the quartz cuvette not inserted fully into the TECAN as…
At some point, when things settle down, I’ll create a page dedicated to the TECAN machine and document all of the troubleshooting and tricks I’ve learned so far. Before that happens I have another mystery to solve. Take a look at the following three scans: This first are some scans of BSA filtrates from…
These volume measures were conducted at 5.6 +/-0.2 PSI using the variable area sepcons with either a half slot, one slot, three slots, or the normal five slots. Note that the pressure was normalized via a pressure regulator, and this normalization was confirmed with a manometer. Filtrates were collected in a series of the…
On Friday Josh and I assembled Sepcon chips with calculated porosity of ~6.5 %, and avg. pore diameter of ~40 nm into Sepcons. I was able to pass 40nm gold through the pores (we don’t have any gold larger than that). Note the “Bad” retentate – for some reason one of the sepcons didn’t filter anything. On…
EDIT: This problem was solved thanks to Tejas. It turns out that Falcon BD transparent 96 well plates absorb in the UV range, and are best used for bradford assays or anything that measures absorbance higher than the UV range. Apparently they sell UV-inactive 96 well plates, but they are very expensive. Josh Winans and…
This week I tried looking into several different ways of detecting the elusive Barcikowski Peptide. We discovered that Coomassie staining does not work, both because of the size of the peptide (it only works on proteins with a MW > 3000Da) and because Coomassie preferentially binds basic residues (and with a sequence of CEEEEEEEEEEEEW, we’re…
Using the remaining 10nm EA-treated chips (all of the 15nm EA-treated and all of the PEG-lyated chips had either been used or broken) I was able to pass cytochrome C through Sepcons. As usual, yields were low (~8-12uL) but the curves were of high quality. The passage of Cytochrome C is not surprising because we…