Diffusion Test– filtered IgG and BSA
The human IgG did not seem to diffuse through the membrane the first time we conducted a diffusion experiment. Barrett believed that the lack of diffusion was most likely due to protein aggregates blocking the pores. So, the human IgG was filtered, and this post is the result of a second round of diffusion experiments.
As a comparison, I set up two diffusion experiments. The first is with hIgG, as in previous experiments. The second is with BSA, to see if the size of hIgG is affecting its ability to diffuse.
Some specs on Wafer 364:
- 2 slots
- average pore size: 26nm
- porosity: 4.69%
- cut-off pore size: 55.52nm
- mean roundness: 0.81 (Standard deviation: 0.12)
Set-up:
- Wafer: 364 (2 chips) (membrane side down)
- 55μL of buffer A in wells
- 7.5μL of 1mg/mL hIgG on top of slits of one chip
- 7.5μL of 1mg/mL BSA on top of slits of other chip
Here are the results from the diffusion experiment (absorption values were found by performing a Bradford assay):
The absorbance reading of the stock proteins are very similar. Looking at the absorbances of the filtrates, BSA has a distinctly higher absorbance reading. After diffusion, hIgG’s absorbance is not much above that of just Buffer A.
To see what possible concentration of hIgG exists in the well after diffusion, I did a standard curve:
are you measuring concentration at 280 or with bradford?