Isolation of an Expressed Product

Introduction
A variety of chromatography techniques have been developed to allow the separation of macromolecules. These techniques take advantage of the differential chemical properties of molecules such as charge, size, and solubility. In the case of green fluorescent protein (GFP), a protein with a number of highly hydrophobic regions, hydrophobic interaction chromatography (HIC) may be used.

First, bacteria containing the protein of interest are lysed, and the extracted supernatant is recovered. A column is prepared containing beads with a hydrophobic surface which will bind other hydrophobic molecules under conditions of high salt (ammonium sulfate) concentration but not at low salt concentrations. The column resin and the extracted sample are equilibrated with high salt and the sample is passed through the column. Hydrophobic proteins bind and others wash through. Then, a low salt solution is passed through the column to elute the hydrophobic GFP.

Part I: Inoculation of Cultures (to be done the afternoon prior to the day of the lab)

Step 1 - For each pair of students, obtain 2 sterile, metal-capped culture tubes from the front table. Pipet 3 ml of LB medium with ampicillin and arabinose into each of the tubes.

Step 2 - Using a sterile inoculating loop (yellow or blue, on front table), touch one of the fluorescing colonies you marked from the previous lab, being careful not to touch any adjacent colonies, and then briefly swirl the end of the loop in one of the tubes of broth. Repeat this procedure for a second colony to inoculate the second tube.

Step 3 - Place the covered cultures in the rack in the 37^oC shaking water bath and shake overnight at 225-250 rpm.

Part II: Isolation of the Green Fluorescent Protein

Step 1 - Select one of the overnight cultures after visualizing under UV light and pour to fill a 2 ml microcentrifuge tube.

Step 2 - Pellet the bacteria by centrifuging for 30 seconds on high speed.

Step 3 - Pour off the supernatant and lightly tap the tube upside down on a paper towel to remove excess supernatant.

Step 4 - Add 250 ul TE/lysozyme (TE w/ 2mg/ml lysozyme) to the tube and resuspend pellet by rapidly pipetting up and down or by vortexing.

Step 5 - Place the tube at -70^oC for 10-15 minutes, remove and thaw to further disrupt the cell wall and membrane.

Step 6 - Pellet the bacterial debris by centrifuging for 10 minutes at high speed.

Step 7 - Meanwhile, prepare the column. Shake vigorously to suspend the resin and then shake down and/or tap on the table to move resin to the bottom. Add 2 ml of Equilibration Buffer, 1 ml at a time. Allow the last 1 ml to drain to just above the resin bed and cap the tube to prevent further flow until the sample is ready to load. (Some students will have columns of a different nature; specific instructions will be provided in lab.)

Step 8 - After centrifugation of the sample, transfer 250 ul of supernatant to a clean tube. Add 250 ul of Binding Buffer and mix.

Step 9 - Elute the remainder of the Equilibration Buffer from Step 7 until the top of the column bed is just dry. Then place the column loosely in a collection tube labeled #1.

Step 10 - Carefully add 250 ul of the sample in Binding Buffer. It is best to let the sample drain down the side of the tube so as not to disturb the top of the bed. Let the entire volume flow into Tube #1.

Step 11 - Transfer the column to a final collection tube labeled #2. Add 250 ul of Wash Buffer and let the volume flow into Tube #2.

Step 12 - Transfer the column to a final collection tube labeled #3. Add 750 ul of TE (Elution Buffer) and let the volume flow into Tube #3.

Step 13 - Examine the samples in each of the three tubes. For this, you may have to spot drops on plastic wrap overlayed on the UV light box or lying under the handheld UV lamp. Record the results. What steps might you take to examine the purity and quantity of your sample?