BIOCHEMISTRY 3723
EXPERIMENT 7
ION EXCHANGE CHROMATOGRAPHY OF GOT
Report Author:
Lab Partner:
Date Due:
Date Turned In:
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Procedural Changes: Indicate any changes you made from the procedure in the lab manual.
Explain why these changes were made and how they affect the results.
Results:
1. Calculate the concentration of protein in Fr. I, Fr. 75%P GOT-1 and GOT-2 using the
Kalb and Bernlohr equation. Show sample calculations and record concentration in the
table below.
[Protein (µg/mL) = 183 A230 – 75.8 A260]
Record this information also in Experiment 9 (Day 1, D and Results I.1.A).
Table I: Protein Concentration of GOT fractions
Fr. I
Fr.
75%
P
GOT
-1
GOT
-2
1st
µL
undil
Fr
dln
µL
H2O
(Exp 6)
dln
factor
1
2nd
µL
dil
Fr
dln
µL
H20
---
---
---
-----
---
---
---
-----
dln
factor
2
Total
Dil.
Factor
A230
A260
dil
conc.
undil
conc.
undil
conc.
Bradford
2. Column Chromatography
a. Record volume of GOT-1 loaded onto column: _____________.
b. Turn in photocopy of chart recording from chromatography (on 8.5x11"paper).
Record the chart speed, the type of column, volume of sample loaded, eluting
solvents and flow rate of column directly on the photocopy. Give the graph a proper
title. Label the axes properly for all plots. Mark the fraction numbers on the x axis as
well as time. Also mark the start of the program. Plot the salt gradient. You can
determine the salt concentration from the time and the program parameters. Prepare a
key for the different plots on the graph.
3. GOT assay of column fractions.
a. Tabulate the volume of sample assayed, the ∆A340/min., and the GOT activity in
(µmol/min.)/mL fraction for each fraction assayed. Show sample calculation for
converting A340/min. to (µmol/min.)/mL.
b. Record the fractions pooled and the total volume. Calculate and record the activity of
GOT-2: Fractions Pooled: _____________. Total Volume: ________________.
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Table 1: GOT activity of GOT-1 and Column Fractions
Fraction
Dialyzed
GOT-1
dln
µL
Assayed

A340
min
GOT activity
(µmol/min)
mL fraction
Fraction
dln
µL
Assayed

A340
min
GOT activity
(µmol/min)
mL fraction
Pooled
GOT-2
c. Plot (bar graph) the GOT activity in each fraction directly on the photocopy of the
chart recording. Label the y axis for GOT activity. How does your activity profile
compare with the A280 profile? Explain how your results do (or do not) agree with
expectations.
d. At what salt concentration does GOT activity elute from the column? Explain what
causes this elution.
e. Record all necessary protein concentration in Results Experiment 9. Do it NOW!
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4. Purification of GOT:
a. Resubmit your (uncorrected) purification table from Exp. 6. Make any necessary
corrections (show work) from original purification table and include that information
in the purification table for this experiment.
b. Using the protein concentration determined in this experiment by the Kalb and
Bernlohr equation, fill in the values for Fraction I and Fraction 75%P on the Exp. 7
purification table. Show needed sample calculations (on back of previous page.)
c. Add to the purification table the information for dialyzed GOT-1 and for GOT-2.
Show calculations as needed on the back of this page.
5. Dialysis
a. What is the total protein in dialyzed GOT-1? in Fr. 75%P (by Kalb and Bernlohr)?
These values should be the same. Why is this so? Calculate the actual % recovery of
total protein from the dialysis procedure. If there has been a significant change in
total protein, explain it.
b. What is the total dialyzed GOT-1 activity? the Fr. 75%P activity determined in Exp.
6? Calculate the % recovery of GOT activity from the dialysis procedure. The
recovery should be near 100% for this step. If the dialyzed GOT-1 activity differs
significantly from the Fr. 75%P activity explain why this is so.
c. Calculate the purification achieved by the ion exchange step only. Discuss whether
the ion exchange chromatography step was a successful purification step.
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d. Compare the total activity of GOT-2 to dialyzed GOT-1. Discuss whether the ion
exchange chromatography step was a good step based on yield. What is the most
likely cause of loss of yield?
6. Fill out the table below. In our experience, the Kalb-Bernlohr equation gives protein
concentrations higher than those determined using the Bradford assay. How closely do
your values agree between the two assays (use the numbers to answer the question)?
How does changing the protein assay affect the apparent specific activity? The
purification? (In theory, although different protein assays may give different [protein]
and thus different specific activities, the fold purification should not differ between
assays) Explain.
Fraction
I
75%P
Bradford
[protein]
K&B
[protein]
Bradford
Sp. Act.
K&B
Sp. Act.
Bradford
Purif
1
K&B
Purif
1
7. The goal of this experiment (see RL Switzer and L Garrity, Experimental Biochemistry,
3rd edition, pp 135-148) is to achieve 80-100 fold purification with ≥ 5% yield. What
results did you get? Comment on how your results compare with this goal.
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