Affinity Chromatography

Affinity
Chromatography
By Grant Akalonu
CHE 447-01
Instructor: Binh Nguyen
Objective/Introduction
The purpose of this experiment was to use affinity chromatography to isolate a lectin protein
from a jack bean meal extract. Affinity chromatography can be used to isolate proteins based on
their affinity for a specific ligand. The chromatography column consists of a matrix which is
covalently bound to a ligand. An extract containing the protein of interest is passed through the
affinity column where the protein ligand interaction takes place. The protein of interest in the
jack bean meal extract used in this experiment is Concanavalin A (Con A). The chromatography
matrix comprising the column contains a glucose based dextran which Con A binds to, so no
additional ligands need to be used for this experiment.
Material/Methods
In a 50 ml tube, 1.5 grams of jack bean meal extract was in 12 ml of 1M NaCl. The Con A
from the jack bean meal extract was extracted for the jack bean meal by holding in a vortex for 10
minutes. The tube was centrifuged at 2000 rpm for for 15 minutes. The supernatant was transferred
to a clean tube and spun again if there was any undissolved jack bean meal remaining. The pellets
were discarded and and 0.5 ml of the extract was saved and labeled as Jack Bean Meal Extract-
Sample 2.
Next was the preparation of the column. Remove the plunger from the syringe. A cheesecloth
was folded in half twice and then again to fit into the barrel of the syringe. Ten ml of slurry was
poured into the affinity column and the gel was allowed to settle. Then the elution of the affinity
column began, as liquid flow from column. The cheesecloth was remove and repacked at the
bottom of the column and repacked. When the affinity gel surface in the column was moist
and no liquid was visible the rest of the column was filled with 20 mL of 1 M NaCl.
To collect samples, the column was charged by gently pouring the extract into the affinity
column. The cap covering the column was removed and the effluent was collected in a clean 50
ml beaker. As the last amount of extract entered the column, the flow was stopped. The affinity
column was washed with 10 mL of 1M NaCl. The column was eluted and the effluent was collected
in a new 50 ml beaker. This was repeated three more times. When the last (fourth) elution wash
entered the column, flow was stopped. This tube was labeled Effluent - Sample #3. Five ml of
elutant, 1M NaCl/1M Dextrose, was poured into the column. Then the column was allowed to flow
and the first 0.5 ml fraction was labeled Eluate fraction 1 - Sample #4. The column flow was
stopped and the column was allowed to set for 10 minutes. After 10 minutes, 8 fractions of 0.5
mL each were collected. The initial fraction collected was labeled as Eluate fraction 5 - Sample
#5 The final fraction as Last eluate fraction collected - Sample #6.
The samples was were then applied to a membrane. Ten L of each sample slowly to the
membrane. The size of each spot was about 10-11 mm in diameter. Then the membrane was
allowed to dry completely for 15 minutes at room temperature.
Results
Fig 1. Membrane Results.
Answers to Required Questions
1) What pattern of enzyme (HRP*) binding activity would you expect if you assayed the 0.5
ml effluent fractions?
If 0.5 ml effluent fractions were used this would lessen the binding activity between enzyme and
substrate. The membrane result would have spots of very light colors, indication that Con A did not bind
to HRP.
2. Dextrose is used to elute the bound Con A from the affinity gel column, yet the bound
dextrose was not removed from the Con A-containing eluate fractions before adsorbing the
fractions to the membrane. Why does Con A bind to HRP* if the dextrose is still present in the
Con A binding site?
Con A binds to HRP because HRP contains mannose. Con A is a mannose binding protein, and binds
strongly to mannose present in HRP than the glucose in dextrose.
3. Con A adsorbs strongly to the membrane, yet the HRP* protein binds only slightly. If HRP
bound as strongly to the membrane as Con A, then the assay may not be possible unless an
intermediate step was done. What would that intermediate step be?
An intermediate step could be coating the membrane with a ligand that Con A does not have an
affinity for. Also, Con A can be eluted out of the membrane with dextrose or another glucose
containing substance. This works best however if the substance does not have an affinity for the
membrane.

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Fig 1. Membrane Results.

Answers to Required Questions

Con A binding site?

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