Protein

Purification by Column
Chromatography
Proteins play key roles in various macromolecular functions in all living organisms.
Catalyzing metabolic processes, acting as structural elements, conveying intra- and
extracellular signals are only three of the many functions they play in the body. To
understand their exact roles and functions we must isolate the proteins and analyze
them using some of the most modern biochemistry procedures.

Column chromatography takes into account the affinity a protein will have to
specific solutions due to their chemical properties. In this instructional set, water
and Hydroxycitric acid (5% HCA) are used to wash away impurities from the
column while pure Methanol (MeOH) will be used to elute the columns to obtain
purified proteins.

Note: There is no single way of purifying protein, this is just one of the possible
purification methods.

Materials

1. Chromatography apparatus
2. Vacuum pump
3. Columns for protein purification
4. Pipette & Pipette tips
5. 2ml Vials
6. 100% MeOH
7. 5% HCA
8. Your protein solutions
9. Paper towels
10. Nitrile Gloves
Columns for
11. Distilled water
protein
purification

Pipette & Pipette tips

Chromatography apparatus

Vacuum Pump

Instructions
Warning: Wear laboratory gloves and coat during entire procedure to prevent any
injuries when handling the solutions

Preparation

1. Obtain your protein samples1 and place near the chromatography apparatus
on your work bench
2. Label a second set of clean 2mL vials, as show in the example below. Be sure
to include at least:
a. Sample name
b. Name of the experiment

3. Place your container of 5% HCA, your container of pure Methanol (MeOH)
and a bottle of distilled water on your bench
4. Set a pipette to 3ml, put a pipette tip on it and place it near the 5% HCA
5. Calibrate a second pipetted to 4ml, put a tip on it and place near the MeOH
6. Set out a few paper towels near the chromatography machine
7. Place chromatography columns near the chromatography machine

Now, you are ready to run your samples and purify your proteins.

Tips to consider before running the chromatography:

o Between steps 6 and 7: Do not let the columns run dry when adding the
HCA after the addition of MeOH
o Achieve this by opening and running the columns one at a time
o In step 8: When loading your protein samples, be sure to keep track of
which sample is in which column
o In step 11: Add distilled water when about 10-15 mm of protein sample
remains in the column
o For step 14: Be sure to place the correct vial under the appropriate
collection spout

1 Crude protein samples are to be pre-prepared in vials and suspended in
appropriate buffer solution

Running the Chromatography

1. Check to make sure every part of the chromatography apparatus is well set
up and in place
2. Place the columns on the spouts of choice located on the top of the
chromatography apparatus

3. Turn vacuum pump on
4. Open turn the pressure knob where the vacuum tube is connected to the
chromatography apparatus, this is allow pressure to build in the
chromatography chamber2
a. Allow pressure to increase to 20-25mmHg
The pressure knob

5. Add 3mL of MeOH to each column
6. Turn open the spout of each column, so that the MeOH in each column runs
through the column and into the chamber
Note: Do not let the column run dry before putting in the 5% HCA
7. Pipette 3mL of 5% HCA into each column
a. Run each column dry and close instantly
8. Load your protein samples to each column
9. Lower the pressure to 5-10 mmHg
10. Open every column and allow the samples to run through3
11. Before the columns run dry, add 1-2 mL of distilled water, this will wash out
any remaining compounds from the column
12. Turn vacuum off and release any pressure left in the chamber
13. Clean out the inside of the chamber with a paper towel4
14. Load your clean and labeled vials to the inside of the chamber
15. Place the top of the chromatography chamber back onto the apparatus
16. Bring inner-chamber pressure back up to 20-25 mmHg

2 Pressure in the chamber is measure in mmHg
3 All columns can be open at the same time since pressure is so low, you will be less
time pressured
4 This will prevent any of the remaining MeOH to wash away the labels on your vials

17. Finally, elute the column with exactly 4 mL of MeOH

Release the chamber pressure, open the apparatus and remove your vials
containing your purified protein samples!