Diffusion Experiment (SC 442)
The results of my last diffusion experiment regarding BSA and filtered IgG (using SC 364) were inconclusive. (See “Diffusion Test– filtered IgG and BSA”) Looking at the histogram of results, there is no clear difference between the absorption of filtered IgG and buffer A.
For this follow up experiment, I used SC 442. Of the available wafers we have pore processed, SC 442 has the second biggest average pore size. SC 442 has an average pore size of 23.5 nm, as opposed to the 26 nm average of SC 364. This difference is with in the margin of error for pore processing. In addition, there may have been other facts preventing the diffusion of IgG through the membrane of SC 364.
SC 442:
- 5 slots
- average pore size: 23.5nm
- porosity: 5.06%
- cut-off pore size: 34.4nm
- mean roundness: 0.83 (Standard deviation: 0.09)
Experiment:
1) Buffer A: 55μl of buffer A in well, 10μl of buffer A on top of membranes
2) BSA: 55μl of buffer A in well, 10μl of 1mg/ml BSA on top
3) filtered IgG: 55μl of buffer A in well, 10μl of 1mg/ml filtered IgG on top
Note: In the previous experiment, the only difference in set up was that I only used 7.5μl on top of the membranes. I could not get the 7.5 μl drop of IgG to cover the five slots, so I had to increase the drop volume to 10μl for this experiment. For consistency, I increased the volume on top of each chip.
Here are the results from the diffusion experiment (absorption values were found by performing a Bradford assay):
The filtrate IgG’s absorption is definitely above the absoption of just buffer A. It is possible that it was a function of the wafer, but it could also be contributed to the increased membrane surface area that came with the increase to 5 slots from 2 slots. In any case, the filtered IgG definitely passed through the membrane.
The next step: Nakul has given me samples from SC 505, which has an average pore diameter of 40nm, which is a marked increase. Hopefully, even more IgG will pass through.
Looks good Carrie. Well done!
Are these measurements of the filtrate or retentate? I’m having trouble understanding the experiment – are you measuring some aspect of membrane separation performance or adsorption of material to the exposed surfaces?
It would be helpful if you could add some type of quick “summary” section that explains what was learned in a simple sentence or two for those of us less familiar with your experiments. Thanks!
The measurements are of the filtrate. This is a preliminary experiment for Harold Smith’s project. If I can get human IgG (a larger protein) to pass through the membrane, then I can say, with relative confidence, that the protein Harold Smith works with will also pass through. The ultimate goal is to separate a small piece of a protein from its larger casing, because the part we want is inactive in the larger complex.
Does that help to better explain it?