Materials and Methods Section
Growth and expression of GFP
First, 10ml of liquid LB media 100ug/ml Amp; 25ug/ml Cam was inoculated with a single bacterial colony of strain G. and grown overnight at 37oC with vigorous shaking until the culture was saturated (turbid). Then 500ml of liquid LB growth media 100ug/ml Amp; 25ug/ml Cam, in a 1 liter baffled flask, pre-warmed to 30oC was inoculated with enough of the saturated overnight culture so the 500ml culture had an OD600 of 0.1 (about 4ml of overnight culture was used). This 500ml culture was then grown at 37oC with vigorous shaking until the OD600 reached 0.5 (more turbid than OD600 0.1). At time zero (OD600 ~0.5), 1ml of the culture was pelleted in a 1.5ml centrifuge tube and the supernatant was discarded. The bacterial pellet was labeled “GO” and stored at -20oC for latter analysis using an SDS-PAGE gel. The culture was “induced” with IPTG (1mM final concentration) and continued to grow. At 3 hours post-induction, 1ml of the culture was pelleted in a 1.5ml centrifuge tube. The supernatant was discarded. The bacterial pellet was labeled “G3” and stored at -20oC for latter analysis using an SDS-PAGE gel. Also, 15ml of the culture was pelleted in a 15ml blue top centrifuge tube. The supernatant was discarded. This pellet was labeled as “G3-15ml” and stored at -20oC to be used latter as the starting material to isolate rGFP.
Preparing the GCE (rGFP Crude Extract):
Set the P1000 to 500ul and added breaking buffer twice (so you end up with 1ml total) into the 15ml conical that contained G3-15ml bacterial pellet breaking buffer-10mM Tris, pH8.0; 150mM NaCl. Then immediately pipette the breaking buffer up and down (using P1000 that is still set to 500ul) until the pellet has thawed and the solution is homogeneous. Keep pipet tip in solution the entire time to avoid introducing air bubbles
Next transfer all of the homogeneous solution into a 1.5ml centrifuge tube, vortex for 5 minutes, label the tube, and place in 37oC water bath for 10 minutes.
While preparing the columns transfer the centrifuge tubes to a rotating platform shaker in a dry air 37oC incubator for 20 minutes to continue the lysing process. After the bacteria have lysed, centrifuge the mixture at 14,000xg, 4oC, for 10 minutes observe and record the fluorescence (using a hand held UV light) seen in the pellet and the supernatant samples.
Then, decant the supernatant to a new centrifuge tube by inverting the tube upside down and avoiding the pellet. This supernatant is called the GCE (rGFP crude extract).
Preparing a Ni+2-agarose column
Assemble column by removing plunger of syringe and adding a luer-lock to the tip of the syringe. Place a small amount of glass wool (about half the size of the end of a Q-tip) into a 3 ml plastic syringe. The glass wool should take up about ¼ ml of the syringe.
Secure the column to a ring stand using a clamp. Make sure column is perpendicular in order to get a good elution profile. Pipet about 100ul of breaking buffer into the top portion of a closed luer-lock so that the breaking buffer is over flowing to insure that no air trapped in the top of the luer-lock. Pipet 1ml of buffer into the syringe column; while the buffer is flowing out the bottom of the syringe column, screw the closed luer-lock onto the syringe.
Add 2ml of additional buffer to the column, open the luer-lock, allow several drops of buffer to flow out of the column, and then close the luer-lock. With at least 500ul of breaking buffer on top of the glass wool, ask the TA to pipet 1ml of a 50% slurry of Ni+2-agarose into the column. Open the luer-lock to “gravity pack” the agarose matrix in the column. Pre-equilibrate the column by adding 5ml of breaking buffer and open the luer-lock to eliminate the ethanol from the Ni+2-agarose slurry from the column then close the luer-lock.
Loading the rGFP sample onto the Ni+2-agarose column
Transfer 100ul of the GCE into a new centrifuge tube and Label this GCE and set it aside for future SDS-PAGE analysis. Then add buffer enough to equal1ml total. With the luer-lock closed, slowly apply the GCE to the Ni+2-agarose column. Wait 5-10 minutes to allow the histidine tag on the rGFP to bind with the Ni+2-agarose matrix. Open the luer-lock and collect ~0.5ml of the effluent into a 1.5ml centrifuge tube labeled W1 (for wash #1) and collect other ~0.5ml in a centrifuge tube labeled W2 and keep luer-lock open for remainder of experiment. Next, observe and record the fluorescence seen in the column before the washing process.
Washing unbound proteins from the Ni+2-agarose column
Proteins that fail to bind the column should come off in the wash fractions.
Wash the column by sequentially gently adding and collecting 0.5ml increments of breaking buffer and allowing all 0.5ml to drain before moving to the next tube. Use a total of 4ml of buffer for this wash step. Label each effluent starting with W3 and ending with W10. Wash the column with an additional 5ml of breaking buffer and discard this effluent.
Eluting rGFP and c-purifying contaminants from the Ni+2-agarose column
Proteins that bound the column should come off in the elution fractions.
Wash the column by sequentially adding and collecting 0.5ml increments of elution buffer 10mM Tris, pH8.0; 150mM NaCl, 300mM imidazole. Use a total of 5ml of elution buffer for this step. The imidazole competes for the Ni+2 binding sites because structurally it looks like histidine. Most of the fluorescence should elute in within the first 1-1.5 ml of applied elution buffer. Label each effluent starting with E1 and ending with E10. Then observe and record the fluorescence seen in the column after the elution process.