DDM Colorimetric Analysis
After talking with Prahnesh, a graduate student in the Dumont Lab I obtained a protocol for a colorimetric assay used to detect the presence of detergents in solution. The assay uses phenol and concentrated sulfuric acid in the following manner:
- add 600uL Sulfuric Acid to 40uL of sample and vortex
- add 200uL 5% Phenol and vortex
- heat at 90-degrees C for 5min
- cool samples to room temperature
- measure absorbance at 490nm
The detection assay works by interacting with the sugar groups associated with the detergent molecules, as those found in DM, DDM, etc. A standard absorbance curve for known detergent concentrations can be constructed and then used to determine the detergent concentrations of unknown samples. In our experimental setup the hopes would be that this assay could be used to detect detergent passing through the membrane, without interacting with proteins such as BSA. Having gathered the necessary chemicals I wanted to test the assays ability to establish a linear relation between absorbance and concentration (%) for the detergent DDM, while also testing its interacting with the BSA protein.
I created dilutions of DDM ranging in concentration from 0.5% to 0.003% (recall the CMC of DDM is 0.009%), as well as dilutions of BSA ranging in concentration from 1.25mg/mL to 0.0006mg/mL. The image above shows the color change in the assay associated with the DDM and BSA.
When the absorbance values were obtained using the TECAN and plotted as a function of solution concentration the following plots were obtained.
The DDM absorbance plot has a clear linear relationship with concentration while BSA absorbance appears to have no relation with concentration. I have not yet tested the absorbance of a mixture of DDM and BSA but from these initial results this assay should allow us to detect any DDM detergent that passes through the membrane without being greatly affected by any BSA that may find its way through.