Biomedical Engineering Reference
In-Depth Information
7.4
Gel Filtration for Further Purification
In order to separate the monomers from dimers, trimmers and multimers, it is im-
portant to further purify the olfactory receptors using conventional gel filtration
method. This is to fractionate dimers, trimers and multimers for subsequent studies.
7.4.1
Materials and Reagents Required for Gel Filtration
1. Amicon Ultra Centrifugal Filter Units with Ultracel membranes (Millipore,
50 kDa MWCO membranes)
2. Sterile 96-well plates with V-shaped bottoms, ~ 500 µl/well capacity
3. Wash buffer: 0.2 % FC14 in DPBS, sterile filtered through 0.22 µm filters
7.4.2
Gel Filtration Chromatography
1. Equilibrate the gel filtration column with at least 1-2 column volumes of wash
buffer. We use a HiLoad 16/60 Superdex 200 column (GE Healthcare) on an
ÄKTA Purifier FPLC system (GE Healthcare).
2. Load the freshly concentrated OR sample into the system.
3. Run the system at 0.3 ml/min, and monitor the UV absorbance at 215 nm and
280 nm. The monomeric form of our receptors typically exits the column at
60-65 ml. We collect the first 40 ml in a clean bottle. The remainder is collected
in four 96-well V-bottom plates with 100 µl in each well.
4. Pool the appropriate eluted protein fractions together.
5. Concentrate the pooled fractions to the desired volume or concentration, and
store them at 4 °C until they are ready for further analysis. Samples can be kept at
− 80 °C for long-term storage and should only be thawed once as repeated freeze-
thaw cycles can induce protein aggregation.
7.5
Notes
1. Brij-35 has been the optimal detergent in our experiments. However, other
groups have found other detergents to be optimal for their GPCRs, especially
other polyoxyethylenes related to Brij-35 [
7
]. A preliminary detergent screen
in which the cell-free reaction volumes are scaled down to a total volume of
25-50 µl may be necessary.
2. The olfactory receptor genes have a 5' NcoI site and a 3' XhoI site for ligation.
They also have a C-terminal bovine rho1D4 epitope tag (TETSQVAPA) for puri-
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