SEPARATION OF BLUE DEXTRAN, CYTOCHROME C AND ...

Lab
501
Introduction
Experiment No. 1
H9SU BIOCHEMISTRY LABORATORY SERIES - Fall 2010
SEPARATION OF BLUE
DEXTRAN, CYTOCHROME C
NAME
SECTION LOCKER
AND VITAMIN B 12 ON
SEPHADEX G-100 AND G-25
INSTRUCTOR DATE
Porous Sephadex gels such as Sephadex G-100,
G-150 and G-200 have found extensive application
for the separation and study of polymers such as
proteins, polysaccharides and nucleic acids.
Sephadex gels are of particular value because
they generally do not destroy the integrity of labile
biological samples.
In experiment No. 1 Blue Dextran 2,000 Mw
2,000,000; cytochrome C, M w 25,000; and Vitamin
B 12,
M.W. 1,357; will be separated on Sephadex
G-100. The eluant will be 0.1 M sodium chloride.
Sephadex G-100 has a molecular weight exclusion
value of 100,000 for dextrans. Therefore, Blue
Dextran 2,000 with an average molecular weight of
2,000,000 will be used to determine the bed void
volume (Vo). The colored compounds will be
detected visually.
Experiment No. 2
Sephadex G-25 has found widespread application
for the rapid, mild and efficient desalting of
polymers such as peptides, proteins, nucleic acids
and polysaccharides. Compounds with molecular
weights of less than 5,000 can also be fractionated
and separated from compounds with molecular
weights greater than 5,000. In Experiment No. 2,
Vitamin B 12 will be separated from a blue dextran
and Cytochrome C. Since the blue dextran and
Cytochrome C with molecular weights of 2,000,000
and 25,000 respectively, both exceed the
exclusion limit of 5,000 for Sephadex G-25, they
will appear as a green band moving in the void
volume. The green band will be used to determine
the void volume of the column.
General Materials
1. Kontes Flexcolumn, 0.7 x 30 cm.
2. 30 small test tubes and rack.
3. Small pipette or dropper
4. Reservoir and connecting tubing.
5. Ring stand and clamps.
6. 25 mL graduated cylinder.
7. Eluant - 0.1 M sodium chloride solution.
8. Dextran - B 12 vial, which contains 9 mg Blue
Dextran 2,000, 9 mg Cytochrome C, and 2.7
mg Vitamin B .
12
9. 0.75 g of Sephadex G-100 Superfine
(Experiment No. 1).
10. 2.5 g of Sephadex G-25 Fine (Experiment No.
2).
11. Fraction Collector
Column Preparation
1. Erect the column with the top cap removed.
Check vertical position with a plumb line or
carpenter’s level.
2. Run a small volume of eluant through the
column so that air bubbles do not remain in or
below the nylon net. Forcing eluant back and
forth through the net with the aid of a syringe
or yellow automatic pipette attached to the
capillary outlet tubing often facilitates this
procedure.
3. Close the stopcock at the bottom of the
column when eluant remains in the column to
a height of approximately 1 cm.
Column Packing and Stabilization*
1. Rapid swelling and packing procedure.
a. Bring approximately 50 mL of eluant (0.1
M NaCl) to boiling.
b. Add 15 mL of boiling eluant to a 25 mL
graduated cylinder.
c. Gradually and with mixing, add the
preweighed gel to the eluant-containing
cylinder.
d. Add additional eluant to give a total

501 - SEPARATION - Page 2
volume of 20-25 mL.
e. Mix gently with a stirring rod for 15
minutes.
f. Allow the gel to settle for 15 minutes.
g. With a dropper remove excess clear
eluant so that a total volume of 18 mL of
gel and eluant remains in the cylinder.
h. Slurry the gel; if bubbles remain below the
surface of the slurry, add slightly more
eluant (2-3 mL).
2. Smoothly pour the gel slurry into the column
until it is full. If air bubbles appear in the bed
and do not rise to the surface, remove the gel
from the column, add 3-4 mL of eluant to the
gel, stir and repour the gel slurry.
3. After 5 minutes the column outlet may be
opened.
4. As space allows, add to the column any gel
slurry remaining in the graduated cylinder.
5. Immediately fill the column with additional 0.1
M NaCl.
6. Adjust the stopcock, so that a flow rate of 0.1-
0.15 mL/min is obtained.
7. Stabilize the bed by washing with eluant for 30
minutes; approximately 3-4.5 mL of eluant
should be collected. A final bed height of
approximately 25 cm should be established.**
Sample Preparation
Dissolve the contents of the Dextran-B 12 vial in 3
mL of hot distilled water. Be sure the sample is
completely dissolved.
Sample Application
1. Remove the column top cap and carefully
remove most of the eluant above the bed
surface with pipette or dropper. WARNING!
DO NOT disturb the bed surface.
2. Let the eluant remaining in the column flow
into the bed. The top of the bed actually
appears to be dry.
3. Close the stopcock.
4. Carefully add 0.3 mL of the sample solution
dropwise to the bed surface. Do not disturb
the surface of the bed.
5. Open the column outlet and begin collecting
the eluant emerging from the column in a
fraction collector set of 10-drop fractions. The
volume of eluant collected from the time the
sample is placed on the bed until the Blue
Dextran 2,000# appears in the eluant is called
the void volume (V o).
To determine this you
will need to measure the volume of the
fractions (average the volumes of several
fractions) and multiply by the number of
fractions.
6. Immediately after all of the sample has flowed
into the bed, carefully add three small portions
(each approximately 4 drops) of eluant to the
top of the bed so that the sample is washed
evenly into the bed.
7. Carefully fill the volume above the bed with
eluant.
8. Elute at a flow rate of approximately 0.1-0.15
mL/min. Adjust the head pressure or
stopcock so that the above flow rate is
obtained.
9. Keep the eluant in the reservoir at a constant
level during the experiment if a Mariotte flask
or similar device is not used.
Collection of Fractions
1. As the Blue Dextran approaches the bottom of
the bed, collect 10-15 drop fractions
(approximately 0.5-0.75 mL each) in the small
numbered test tubes. Be sure to record the
volume of clear eluant collected in the
graduated cylinder or by the technique
described above for the V o determination.
2. Collect approximately 25 fractions. Collect
two fractions after the last component is no
longer visible in the collected fractions.
3. Assign a number value from one to four to the
tubes containing colored components which
corresponds to the relative intensity of the
color in each tube. Tubes which are colorless
assign a value of zero.
4. Record the assigned number for color
intensity.
5. Measure the total volume in which each
component is eluted from the column. Record
this information for later calculation of dilution
factors.
6. Mark the top of the bed by placing a piece of
tape on the outside of the column. Empty the

Calculations
column of gel. Fill the column with water
to the tape mark and measure the volume
in a graduated cylinder. This volume is
equivalent to the bed volume (V t ) . Record
this volume.
1. In each experiment plot the relative intensity of
the color of each component versus the
corresponding fraction number of graph paper.
2. The elution volume for a component
represents the volume of effluent collected
from the time the sample is placed on the
column bed to the time the component
appears in the effluent. Consider this point to
be the effluent volume to the fraction where
the component first shows its half maximum
color intensity.
3. Estimate the efficiency of separation from the
graph. (See the booklet in lab.)
4. Calculate dilution factors for the components.
5. Calculate V i. Then calculate the Kd and Kav
values for the components. Assume that the
elution volume of Blue Dextran 2,000 is equal
to the void volume.
*NOTE. The packing and stabilization procedures
used in these experiments are designed so that
the experiment can be completed with a 3 hour
laboratory period. For more exacting work, gel
swelling, bed packing and stabilization procedures
described in Sephadex technical data sheets and
the booklet, “Sephadex-Gel Filtration in Theory
and Practice,” should be employed.
**Longer beds may be used for greater resolution
and should be packed by the procedure described
in the booklet. “Sephadex-Gel Filtration in Theory
and Practice.”
#See Technical Data Sheet No. 8.
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