Download Lab Activity 1

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Dialysis
• Passage of solutes through a semi-permeable
membrane.
• Pores in the dialysis membrane are of a
certain size.
• Protein stays in; water, salts, protein
fragments, and other molecules smaller than
the pore size pass through.
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When is dialysis recommended?
1. If working with proteins
and nucleic acids (elimination
of small molecular weight substances
such as reducing agents such as DTT,
BME, labeling reagents or
preservatives (e.g. sodium azide).
2. Exchanging buffers for
electrophoresis, ion
exchange or affinity
chromatography.
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Dialysis
• Following a salting-out step, the solution will contain
a high concentration of salt that can be disruptive to
subsequent chromatographic steps.
• The salt can be removed by dialysis – dialysis tubing
has pores with a specific molecular weight cut-off that
allows smaller molecules (salt) to pass.
Buffer– large volume
Dialysis tubing with protein and high salt
Exchange buffer
> 3 times
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Experiment
• Reagents
1. Saturated solution of Ammonium Sulfate
2. 5% aqueous solution of barium chloride
3. Biuret reagent
4. Egg white ( an aqueous solution) or blood
serum sample
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Materials
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Cellophane cut into sheets of 12.5 x 12.5 cm
A beaker with distilled water
Glass rods
Small rubber rings
A test tube stand with a set of test tubes
pipettes
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Procedure..
1. To 5 ml of egg white soln ( or blood serum) in a test tube,
add a couple drops of sat., (NH4)2SO4, then stir with a glass
rod.
2. Sample 10 drops of the mixture into 2 test tubes; use them
to perform the biuret test and the SO42- test.
3. To perform the test for sulfate ion, add 2 to 3 drops of
barium chloride.
4. Shape the cellophane sheet into a sack, then fill it up to 1/3
of its volume with the protein solution.
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Procedure..
5. Clip the sack edges between 2 glass rods tightly held against
each other with rubber rings that clasp the glass rods at their
ends.
6. Place the cellophane sack into distilled water, so as to keep the
sack submerged in water. The level of protein solution in sack
must be below the water level in the beaker.
7. Let the dialysis proceeds for about 1 h. then take 2 samples
(10 drops each) of the liquid contained in the beaker (dialyzer)
to perform the biuret test for protein & sulfate ion.
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