Biochemistry and Molecular Biology

Experiment Report Sheet

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 SHANDONG FIRST MEDICAL UNIVERSITY

Separation and purification of serum γ-globulins

1. Principle

Serum proteins are classified for two kinds by electrophoresis, that is albumin and
globulins (α-, β-, γ-globulins). γ-globulins including immunoglobulin are similar
protein family in primary structure and function. Therefore, isolation and purification
of functional γ-globulins from serum is an important step for medicine research.
There are many methods for protein separation based on their difference in
solubility, charge, molecular size and affinity for a ligand. However, there is no any
single method to separate the given protein from cells or tissue. Combination of
several methods is usually used in protein isolation.
This experiment introduces the procedures for separating γ-globulins from serum
by and . The addition of saturated
(NH4)2SO4 to protein solution destroying And of protein
resulting in its precipitation, which is referred as salt precipitation. Since the particle
size and hydrophilic extent are different, the amount of salt addition varied for
different protein precipitation. The serum globulins are , but albumin
are soluble in 50% saturated (NH4)2SO4. However, saturated (NH4)2SO4
is enough for γ-globulins precipitation. After two centrifugations, the precipitation
containing rough γ-globulins is dissolved in PBS. The rough extracts contains
considerable amount of (NH4)2SO4, and usually, it must be removed before further
research. Gel filtration is a liquid chromatographic method of separating solute
molecules based on difference in molecular size. When proteins and (NH 4)2SO4 are
passed down a column containing very small porous beads of a highly hydrated
polymer, can penetrate into the pores of the beads and thus retarded
in their flow down the column, but can not penetrate the pore and
emerge from the column first.

2. Materials

2.1 serum
2.2 saturated (NH4)2SO4
2.3 phosphate buffer saline (PBS)
2.4 sephadex G-100
2.5 Biuret reagent
2.6 Nye’s reagent

3. Procedures

3.1 Salting out

3.1.1 pipette ml serum, add ml PBS and mix completely. Add ml
saturated (NH4)2SO4 with simultaneous stirring and mixing.
3.1.2 stand for 10 min at room temperature, then centrifuge at rpm for
10min.
3.1.3 remove and discard the supernatant. The precipitation is dissolved in ml
PBS, and then ml (NH4)2SO4 is added. After complete mixture, stand for
10 min at room temperature.
3.1.4 centrifuge as 3.1.2.
3.1.5 discard the supernatant and the pellet is dissolved in 0.2 ml PBS.
3.2 Desalting
3.2.1 packing the column
① place the glass column vertically in chromatographic rack
② fill the column with PBS for checking leaks and then open the clamp for empting
the PBS.
③ with the column about one third full of PBS, pour the swollen Sephadex G
into column with continuous stirring of the gel.
④ open the clamp and adjust the flow rate to about drops/ min.
3. 3 sample application
① allow the PBS to drain to the gel surface. Until the surface of the bed is just barely
dry, apply the sample gently on the gel bed, let all samples run into the surface and
then add more PBS buffer for a continuous elution.
② discard 20 drops and then collect the elution in a glass tube, 10 drops each tube.
3.4 detection
3.4.1 protein detection
① Add two drops of collected elution into one nest of china plate, drops the same
amount Biuret reagent in the nest and mix completely.
② Observe the color and record the result. “+” represents positive (more “+”, higher
content of proteins), “-” means negative.
As soon as protein emerges from the column, the color shifts from blue to .
3.4.2 (NH4)2SO4 detection
Detect (NH4)2SO4 with Nye’s reagent. As protein detection, please Observe the
color carefully and record the result. When (NH4)2SO4 flows down from the column,
the color will change to .

4. Results

4.1 please display your results with tables ( “+” represents positive; “-” means
negative)
Protein results
Tubes No.

results
(NH4)2SO4 results

Tubes No.
results

4.2 please draw the separation curve according to your results (tube number as X
axis; “+” as Y axis).