1. Protein Production Jackpot!
GFP column chromatography

2. Post pGLO: Now you have a protein but…. How do you use it?
How would you be able to test just the GFP protein?
How do you verify your results?

3. THIS IS WHERE WE WILL START!! Final spin and Prepping the column
Thaw the tube by holding in your hands for warmth…
Spin for 10 minutes
Prep the column during the spin cycle
Remove the cap on the top and bottom
Add 2 mL Equilibrium buffer to the top of the column (High Salt Buffer!)
Complete 2 separate 1 mL aliquots
Drain to the 1 mL mark on the column (You might want to mark with line with a sharpie before you start.
Equilibrium buffer is very high salt. This changes the shape of the protein. It causes the hydrophobic regions to move outward and the hydrophilic regions inwards. Since GFP has lots of Hydrophobic amino acids. It kind of turns it into a big sticker, so it can be easily caught in the column.

4. What will we do….
Several Day process, we will condense into 45 minutes!!!
Separates the GFP protein created by the transformed bacteria in pGLO, from the rest of the proteins in the E.Coli.
GFP has lots of hydrophobic amino acids… we use that to help it stick to the column
Wash out all the hydrophilic proteins
And finally wash out the GFP and collect it in the final tube.

5. Mass Production!!
Label 2 prepared culture tubes, + and –
Using a sterile loop for each, remove one of the whitish colonies from each plate.
LB/amp/ara into the + tube
LB/amp into the - tube
Immerse the loop with the colony into the culture medium
spin to disperse
Cap and Shake! Plenty of air!
Incubate overnight or for a couple of days.
I’ve done this for you!! You can definitely do it with your students!
This is a good place to stretch a kit if you need too…. You can skip the minus tube, it is simple a control. You still only have 8 columns provided in the kit, that is your limiting nutrient.
This can be done on the last day of the pGLO lab when you are checking results OR… you can wait , store plates in the fridge and complete this later.

6. Concentrate! Label a micro tube, +
Observe your + culture tube with the UV light, record your observations
Aliquot 2 mL of the culture broth into the micro tube.
Place the micro tube into the centrifuge and spin on high for 5 minutes.
Rinse your pipette with distilled water after each use.
Removing everything but the bacteria. By spinning the sample we are forcing all the bacterial cells to move towards the end of the micro centrifuge tube. This creates a concentrated pellet of bacterial cells.
I’ve done this for you!! You can definitely do it with your students!

7. Draining…
Pour all the liquid out of the top of the centrifuge tube.
Observe the pellet at the bottom of the tube with the UV light.
Using the rinsed pipette, add 250 ul of TE buffer.
Move the liquid up and down in the tube using the pipette to resuspend the bacteria.
We are removing any remaining nutrient broth and stabilizing the sample with the buffer. The buffer helps to control pH and prevent any degradation of the sample during the following processes.
I’ve done this for you!! You can definitely do it with your students!

8. Digestion Add 1 Drop of Lysozyme to your tube.
Cap and gently mix by flicking a few times.
Place your sample in the freezer overnight.
The lysozyme helps to start digesting the proteins of the cell walls. This will release the protein that is currently trapped inside the bacterial cell walls. Think of this like a glass bottle full of water, if we put it in the freezer and the water expands we will break the jar…. Same thing here, it helps to release everything inside the bacterial cells including the GFP!
I’ve done this for you!! You can definitely do it with your students!

9. Recap! Let the separation Begin!
What
How
Why
Status 1.
Mass produced bacteria that create GFP protein
Grew transformed bacteria in culture tubes
Increase amount of the desired protein
DONE 2.
Broken Down the cells walls
Enzymatically- Lysozyme
Physically- Freezing overnight
Releases all the proteins from inside the bacterial cell walls. 3.
Collect the protein
Column chromatography, Trap the GFP in the column, wash out everything else, then wash out the GFP
Separates GFP (the desired protein) from all the other proteins created by the cells.
NOT YET!

10. Binding the Protein
Using a NEW pipette add 250 ul of the supernatant (Liquid) into a NEW micro tube.
Add 250 ul of Binding Buffer (BB) to the micro tube with the sample.
Cap and gently flick with your finger to mix.
Everything that is solid is now trash! It liked a cracked egg and all that is left is the shells. The Binding buffer has very high salt content causes the GFP to be trapped in the column.

11. Let the Drain begin. Label 3 collection tubes 1,2,3
Place in the foam rack
Remove the cap from the column and place into #1 tube
Let the buffer drain to the top of the matrix.
Add a little pressure to speed up this process:
Draw up the plunger on the syringe
Place the stopper in the top of the column
Gently push some air through the syringe until you reach right up the matrix.

12. Loading the sample
Using a NEW pipette aliquot 250 uL sample from the micro tube into the column
SLOWLY add by touching the tip to the side of the column and dripping it down into the column
Let it continue to drain into tube 1 until it stops dripping.
Add pressure to speed up the process. Stop when the liquid reaches the top of the matrix.

13. Sorting the trash… Switch to tube #2.
Using a new pipette, Add 250 uL of Wash Buffer to the column
Let the entire amount flow through the column
Add pressure as needed.
Wait for it to stop dripping!
The wash buffer rinses out all the hydrophobic proteins. Everything collected here is TRASH!

14. The Final Countdown Switch to tube #3
Using a new Pipette, Add 750 uL of TE buffer
Let the entire volume flow through the column
Observe using UV light, watch closely as the GFP moves down the column.
Cap all your collection tubes
The last buffer has low salt levels… The hydrophobic areas of the protein move back inwards. it causes those stickers to get pulled back inside and the GFP is released from the column.

15. Results! Most of the GFP should have released from the column…
It will be collected at the bottom of tube 3!
The other tubes contain left over buffer solutions as well as other proteins from the lysed bacterial cells.