MBB 130 Molecular Biophysics

EXPERIMENT 4
Purification of proteins through metal affinity chromatography

This experiment will allow students to experience the purification of a recombinant protein previously expressed in a bacterial
expression system. The purification will involve immobilized metal affinity chromatography (IMAC) using the BD tm Talon IMAC
resin.

Pre lab questions

1. What is the molecular weight of the FP expressed from the plasmid? Give background information on the expressed FP.
2. What methods may be used to purify expressed recombinant protein?
3. What is the basis of separation for the column used in this experiment?

Methodology

Day 1: Purification
1. Pipette 1 mL of the TalonTM resin suspension into the column with the end-cap in place. Allow the resin to settle out of
suspension. This will give you roughly 0.5 mL bed volume of resin.
2. Remove the end cap and allow the suspension buffer to drain until it reaches the top of the resin bed. Make sure the
resin is not dried out and that no air bubbles are trapped in the resin bed.
3. Add 10 bed volumes of 1x Equilibration/Wash Buffer in order to pre-equilibrate the resin. Allow the buffer to drain out
and repeat with another 10 bed volumes. Add the clarified sample. Gently agitate the resin for 20 mins, RT.
4. Allow the clarified sample to elute out of the column and collect it as the “flowthrough”.
5. Add 10 bed volumes of 1x Equilibration/Wash Buffer. Gently agitate for 10 mins, RT. Collect this as the “wash”
fraction and repeat with another 10 bed volumes.
6. Add 5 bed volumes of the Elution Buffer. Collect the eluate in 500-µL fractions.
7. Wash the resin with 5 bed volumes of 20 mM MES Buffer (pH 5.0) containing 0.1 M NaCl.
8. Rinse resin with 5 bed volumes of sddH2O.
9. Add 2 mL of 20% EtOH with 0.1% azide. Allow 1 mL to flow out of the column. Store the column at 4 oC.

Day 1: PAGE
1. Take two 10-ul aliquots from each collected fraction and transfer into 0.2 ul PCR tubes. Add 10 ul of SDS PAGE
treatment buffer to each sample.
2. Boil one the tubes per sample.
3. Load both boiled and not boiled samples into an SDS PAGE gel.
4. Set the electrophoresis at constant voltage (120 V) and constant current (25 mA). Run the gel until the leading dye has
reached the lower end of the gel.
5. Stain the gels with Coomassie.

Day 2: PAGE destaining and gel drying

Post lab questions

1. Describe the process through which IMAC works to purify proteins.

2. Define the following terms associated with column chromatography:
a. Flowthrough
b. Wash fraction
c. Elution fraction
d. Strip fraction
3. Was the FP sufficiently purified by the techniques used? How were you able to determine this?