I performed a DEAE-cellulose column prior to mmp9 purification to remove phenol
ID: 217233 • Letter: I
Question
I performed a DEAE-cellulose column prior to mmp9 purification to remove phenol red. We used 2 buffers Equilibration buffer and a TNC buffer. I'm really confused with the whole prcoess. How I understood was the column was equilibrated with the equilibration buffer and then the media was added then phenol red interacts with the column and the effluent is our protein of interest. When do we use the TNC buffer? And please describe the procedure with chemistry behind. Like what gets trapped and stuff
Buffers
NaCl-Free DEAE-Cellulose column equilibration buffer
10mM Tris-HCl pH 8.0
10mM CaCl2
0.05% (vol/vol) Brij 35
Tris-NaCl-CaCl2 (TNC buffer)
50mM Tris-HCl pH 7.5 4°C
0.15M NaCl
10mM CaCl2
Explanation / Answer
Diethylaminoethyl cellulose (DEAE-C) is a positively charged resin used in ion-exchange chromatography, a type of column chromatography, for the separation and purification of proteins and nucleic acids. Gel matrix beads are derivatized with diethylaminoethanol (DEAE) and lock negatively charged proteins or nucleic acids into the matrix. The proteins are released from the resin by increasing the salt concentration of the solvent or changing the pH of the solution as to change the charge on the protein.
In ion exchange chromatography, the stationary phase is first equilibrated with a low ionic strength buffer. The protein sample is then loaded onto the stationary phase in the same low ionic strength buffer used for equilibration. The bound protein is washed extensively before elution with a higher ionic strength buffer or, in some cases, a buffer with altered pH.
TNC buffer is routinely used as though the exact protein concentration is not critical, but DARTS (drug affinity responsive target stability) may not work as well if the protein is too dilute.