Begin by thawing the frozen solute carriers or SLC cell pellets in a water bath at room temperature. Prepare solubilization buffer by combining 135 milliliters of base buffer and three protease inhibitor cocktail tablets. Once the tablets are dissolved, resuspend the pellet in the solubilization buffer.
Then add deaminase and transfer the suspension into an ice cooled down homogenizer. Homogenize the solution by moving the plunger up and down approximately 20 times, keeping the homogenizer on ice. Next, add detergent stock solution to 1%final concentration.
Transfer the solubilization mixture to a 50 milliliter conical tube and rotate slowly at four degrees Celsius. After one hour, centrifuge the mixture, and collect the supernatant. Equilibrate a four to six milliliter bed volume of Strep-Tactin resin with the base buffer.
Then add equilibrated resin to the solubilized supernatant, and rotate for two hours at four degrees Celsius. Pour the solution onto a gravity flow column and allow the solution to flow through. Wash the resin with 30 times the bed volume of Strep Wash Buffer in three equal steps.
Next, add three to five milliliters of illusion buffer and incubate for 15 minutes before collecting the elute. Measure protein concentration by UV absorbent spectroscopy. Combine the desired illusion fractions, and add 3C protease.
Rotate the tube slowly overnight at four degrees Celsius. The next day, equilibrate two to four milliliters of bed volume of cobalt metal affinity resin with SEC buffer. Add the equilibrated cobalt metal affinity resin to the overnight 3C reaction mixture, and rotate at four degrees Celsius.
After one hour, pour the solution into a gravity flow column and collect the flow through. Concentrate the flow through in a 100 kilodalton cutoff centrifugal filter by spinning at 3000g at four degrees Celsius. Equilibrated dextran argos based SEC column with SEC buffer.
Inject the sample into the sample loop and run the SEC program with a flow rate, such that column pressure is below the column manufacturer's specifications. Using a fraction collector, collect 300 microliter fractions over the entire SEC run. After pooling peak fractions, measure the absorbance at 280 nanometers.
Then concentrate the samples to the required volume in a 100 kilodalton cutoff filter. Initial small scale SLC protein expression was analyzed by SDS page electrophoresis. Fluorescence microscopy of A GFP tagged SLC, confirmed the protein localization, specific to the plasma membrane with significant intracellular accumulation.
Chemically and structurally homogeneous protein yielded a single mono disperse A280 peak during size exclusion chromatography.
