Affinity Chromatography GeNeiTM Affinity Chromatography GeNeiTM

GeNeiTM Affinity
Chromatography
Teaching Kit
Manual

Cat No. 6104100011730

Revision No.: 00050516

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Affinity Chromatography GeNeiTM Affinity Chromatography GeNeiTM

CONTENTS

Page No.

v Objective 1

v Principle 1

v Kit Description 3

v Materials Provided 4

v Procedure 5

v Result 10

v Ordering Information 11

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Objective:
To learn the technique of protein purification by affinity
chromatography by performing the following experiments:
• Purification of Horse Radish Peroxidase (HRP) using
Concanavalin A Agarose column (Con-A).
• Estimation of HRP activity.
• Estimation of Protein concentration by Lowry’s
method.

Principle:
Affinity chromatography is a method of selectively and
reversibly binding proteins to a solid support matrix based on
the fact that biological affinities exist between molecules,
e.g., antigen with antibody. One of the components, the ligand
is immobilized on a solid matrix, which is then used to
selectively purify the target protein. By Including a competing
ligand in the mobile phase or by changing the pH, the target
protein is eluted.

The performance of affinity chromatography is determined

by comparing the specific activity of the protein before and
after affinity purification. The Specific activity of the protein
(enzyme) is defined as its activity per mg of protein. In order
to determine the specific activity, enzyme activity and protein
concentration are determined.

Lectins are plant proteins that have high affinity for particular
sugars such as those present in glycoproteins. HRP is a
glycoprotein, which helps in its selective binding to the
column. Pure protein will then be eluted out using a free
sugar (eg., fructose) that competes with the glycoproteins
for the immobilized lectin.

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Affinity Chromatography GeNeiTM
HRP acts on hydrogen peroxide to release nascent oxygen
that oxidizes ABTS to give a coloured product. The intensity
of the coloured product is measured at 725 nm using a
spectrophotometer or in a colorimeter using a suitable filter.
Protein Estimation by Lowry’s method: This method is
based on both Biuret reaction and Folin-Ciocalteau reaction.
Here, the peptide bonds of protein react with copper under
alkaline conditions producing Cu+, which reacts with Folin’s
reagent to give blue colour due to the reduction of
phosphomolybdotungstate to heteropolymolybdenum. The
intensity of the colour is dependent on the Tyrosine and
Tryptophan content of the proteins.
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Affinity Chromatography GeNeiTM
Kit Description:
Using this kit, students will carry out affinity purification
of Horse Radish Peroxidase (HRP) from crude extract using
a lectin column (Con A).
Students will estimate the HRP activity using ABTS and
and protein concentration of samples before and after
purification to determine the specific activity of HRP.
Enzyme activity of HRP will be determined using ABTS
[2,2’-azino-bis (3-ethylbenzthiazoline)-6-sulfonic acid] as
substrate.
Enzyme HRP activity is expressed in ABTS units, which
is defined as the amount of HRP that oxidizes 1 µmol of
ABTS in 1 minute to ABTS oxide.
From the specific activity of HRP, before and after
purification, the yield and fold purification will also be
determined.
6104100011730 : The kit is designed to carry out 5 protein
purification experiments by affinity
chromatography.
Duration of experiment: Approximately 3 hours
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Materials Provided: Note:

The list below provides information about the materials • Read the entire procedure carefully prior to starting
supplied in the kit. The components should be stored as the experiment
suggested. • Concanavalin A-Agarose suspension cannot be reused.
• Prepare substrate solution for determining HRP activity
Quantity just before use.
• Resuspend a single aliquot of crude sample and BSA
Materials 6104100011730 Store (Protein Standard) per experiment, before use.
(5 Expts.) • Prepare Complex Forming Reagent by mixing one
ABTS 50 ml 4°C volume of Solution I with 100 volumes of Solution II,
just before use.
Column 1 No 4°C • A Single column is provided, this should be washed
Concanavalin A- Agarose 4°C with distilled water before each use.
Suspension 2.5 ml
Procedure:
Crude Sample 5 Nos. 4°C
Purification of HRP using Con A :
Elution Buffer 5 ml 4°C
1. Resuspend an aliquot of Crude Sample in 1 ml of
Hydrogen Peroxide 0.1 ml 4°C Sodium Acetate Buffer.
Sodium Acetate Buffer 60 ml 4°C 2. Fix the column to an appropriate stand.
3. Gently pipette 0.5 ml of Concanavalin A-Agarose
BSA (Protein Standard) 5 x 1 mg 4°C
Suspension into the column provided and allow the resin
Solution I 1.5 ml 4°C to settle.
Solution II 100 ml 4°C 4. Equilibrate Con A-Agarose Column with 5 ml of Sodium
Acetate Buffer. Allow the buffer to drain out completely.
Solution III 10 ml 4°C
5. Save 0.1 ml of the crude sample for determination of
enzyme activity and protein concentration.
6. Load the remaining 0.9 ml of the Crude Sample on the
Materials Required but not provided:
column. Allow it to drain off completely.
Equipment : Colorimeter or Spectrophotometer. 7. Wash the Column with 1 ml of Sodium Acetate Buffer.
Glassware : Measuring cylinder, Test tubes. Add another 4 ml of the Buffer to wash out any unbound
Reagent : Distilled water. sample.
Other Requirements : Glass cuvette, Micropipette, 8. Elute the bound HRP by using 1 ml of Elution Buffer.
Micro Tips and Column Stand. Collect the entire amount in a test tube. Label this
sample as ‘Eluate’.
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Estimation of HRP activity: where: (dA-725) – change in absorbance at 725 nm after

1. Prepare substrate solution by adding 2 µl of Hydrogen
peroxide (H2O2) to 10 ml of ABTS solution.
2. Using the substrate solution, blank the
spectrophotometer/colorimeter reading to zero at
725 nm.
3. To 2.99 ml of substrate solution, add 10 µl of ‘Eluate’
(step 8 of purification) and mix. Note down the change
in absorbance at 725 nm, exactly after 20 seconds of
mixing the reagents refer to this as dA-725 of eluate.
4. Dilute the crude sample (Step 5 of purification) 1:20
(10 µl of crude sample + 190 µl of distilled water).
5. To 2.99 ml of substrate solution add 10 µl of 1:20 diluted
crude sample and mix. Note down the change in
absorbance of crude sample at 725 nm exactly after
20 seconds of mixing the reagents. Refer to this as
dA-725 of crude sample.
6. Estimate the activity of enzyme in both the crude and
eluted samples, as follows:
Extinction coefficient of ABTS at a concentration of
1 µmol/µl = 19.0
Activity of enzyme in the reaction mixture
=
3 x (dA-725)
19
=
__X__ µmol / min.
Therefore, activity of enzyme in the sample
=
X x rv x df
sv
= _____ U / ml
20 seconds
U - ABTS units.
rv - reaction volume (in ml).
sv - sample volume taken for assay (in ml).
df - dilution factor. Where df = 1 for Eluate and 20 for crude
sample.
7. Estimate the total enzyme activity in crude and Eluate
samples as follows:
Total enzyme activity in crude sample
= activity/ml x volume loaded on the column
Total enzyme activity in eluate
= activity/ml x eluted volume from the column
Estimation of Protein concentration by Lowry’s Method:
1. Dissolve 1 aliquot of BSA (Protein Standard) in 1 ml of
distilled water to get stock concentration of 1 mg/ml.
2. Use 1:20 diluted crude sample (Step 4 estimation of
HRP activity) for estimation of protein concentration.
Do not dilute the Eluate (Step 8 of purification) for protein
estimation.
3. Pipette BSA Protein Standard solution and samples
(Crude and Eluate) as given in table I and adjust the
volume to 0.1 ml with distilled water.
4. Add 2 ml of Complex Forming Reagent to each tube,
mix and incubate at room temperature (RT) for
10 minutes.
5. Add 0.2 ml of Solution III, mix and incubate for
20 minutes at RT.
6. Read the optical density using a spectrophotometer at
660 nm or in a colorimeter using a suitable filter. Record
your results as in table 1.

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Table 1: Dilution of BSA (Protein Standard) and Samples. Estimation of Specific Activity of HRP:
Calculate the specific activity using the following formula for
Sl. Standard Amount Water OD Reading
both eluted and crude samples.
No. (in µg) (in µl) (in µl) (A660)
Specific activity = Enzyme activity
1 Blank 0 100 Protein concn.
2 10 10 90 ______ µmol/min/mg
=
3 20 20 80
Estimation of yield and recovery of HRP:
4 40 40 60
The following formula is used to estimate the percentage of
5 80 80 20 enzyme activity recovered on loading the crude sample on
the column:
6 Eluate 25 75
7 Crude Sample Recovery = Total activity in eluate x 100
(1:20) 15 85 Total activity in crude sample
8 Crude Sample Yield = Total activity of eluate/ml x Total volume of eluate
(1:20) 25 75
= ______ U
7. Construct a calibration curve by plotting optical density
reading on ‘Y’ axis against Standard Protein Estimation of Fold Purification:
concentration in µg on X axis. Fold purification = Specific activity of eluate
8. Record the value ‘X’ from the graph corresponding to the Specific activity of crude sample
optical density reading for Eluted and Crude samples.
9. Calculate the concentration of protein in these samples This is a measure of the efficiency of purification of the
using the following formula: enzyme using affinity chromatography.

Sample concentration = (X x df) = ____mg/ml

v

where: X - Value from graph in µg

v - Volume of sample in µl.
df - dilution factor of sample (if any)
(df = 1 for Eluate; 20 for Crude Sample)

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Result Ordering Information

Record your results as shown in Table 2:
Product Size Cat #
Table 2:
GeNei™ Affinity 1 EA 6104100011730
Assay Crude Sample Eluate
Chromatography Teaching Kit
Enzyme activity (U/ml) (Consumables for 5 experiments)

Protein concentration (mg/ml)

Specific activity (U/mg)
Email:
From the above data, calculate and report recovery, yield Sales:
and fold purification of the enzyme HRP. sales@geneilabs.com

Customer Support:
techsupport@geneilabs.com

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Notes:

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