Plasma proteins
The major proteins present in plasma are of 3 groups:
1. Albumin: 36-50 g/l
2. Globulin: 18-32 g/l
3. Fibrinogen: 2-4 g/l
In this test the 3 main plasma proteins will separated from the plasma and
fractioned by means of the salting out technique.
The separated fractions of the proteins will be passed through a gel- column
individually for the purpose of determining its molecular weight.
Last, the separated fractions will be run under current through an SDS- gel
electrophoresis also for m.w determination.
I: Salting out:
1. Blood will be withdrawn from rabbits by syringes containing an anti
coagulant ( usually EDTA is used), the blood then will be placed in
centrifuge tubes and centrifuged for 10 minutes at 3000 rpm to
separate the plasma.
2. Plasma will be collected and the volume is measured.
3. The weight of ammonium sulphate needed to reach 30 % saturation is
calculated from the table provided ( note: correct for the plasma
volume collected)
4. Ammonium sulphate should be weighed carefully and added slowly to
the plasma with constant stirring.
5. This should be centrifuged, collect the supernatent and measure its
volume, the precipitate should be dissolved in 5 ml phosphate buffer
and labeled as F1 ( containing Fibrinogen)
6. The amount of ammonium sulphate needed to bring the solution to
55 % saturation is calculated and added to the supernatent as done in
the previous step.
7. The second centrifuge will take place, the precipitate should be
dissolved in 5 ml phosphate buffer and labeled as F2 ( containing
Globulin), the supernatent will be labeled as F3 which contains
albumin.
Materials:
1. Solid ammonium sulphate.
2. Phosphate buffer PH 7.2 ( take 7.098 g of Na2HPO4 + 5.999 g of
NaH2PO4 and make the volume up to 2 L with distilled water.
3. Centrifuge and centrifuge tubes( plastic)
4. EDTA
5. Syringes.
6. Scissors and cotton
7. Aluminium foil
8. Beaker with glass rod
9. balance
10. Filter paper.
11. A rabbit
II: Chromatography:
The method used here is gel chromatography with the main objective of
measuring the molecular weight of the separated proteins (F1, F2, F3 )
Method:
1. The gel should be soaked in phosphate buffer for at least 24 hours( 6 g
of gel in 300 ml buffer)
2. The gel should be boiled, cooled at room temperature then the column
should be packed with the previously soaked gel.
3. The gel should settle for at least an hour before applying the samples
with the out let closed.
4. A sample of blue dextran should be added first, 1.0 ml of the dye will
be added at the top of the gel after carefully removing the excess
buffer, with a Pasteur pipette the dye will be added carefully avoiding
to disturb the gel bed.
5. Open the out let after 2 minutes to make sure the sample emerged
into the gel then start collecting 1 ml elute with the continues addition
of buffer at the top of the column ( you have to make sure the gel is
never dry ).
6. 20-25 samples has to be collected, add to each tube 1.0 ml of buffer.
Read the absorbance against a blank buffer at 570 nm using plastic
cuvettes.
7. Draw a curve of absorbance against fraction # and obtain the highest
point from the graph this will be the void volume to be used in your
calculations ( Refer to lab sheet )
8. Repeat the same steps for each of the protein fractions but with
measuring the absorbance at 280 nm with the use of quartz cuvettes.
Materials:
1. Sephadex G-100
2. Blue dextran
3. Phosphate buffer
4. Plastic cuvettes
5. Quartz cuvettes
6. Spectrophotometer
7. Test tubes
Electrophoresis:
SDS- polyacrylamide gel electrophoresis will be applied on the protein
samples
Chemicals needed:
1. 1M Tris- 1M Glycine: Take 12.114g of tris + 7.507g of glycine,
dissolve in little water then make up the volume to 100 ml by distilled
water.
2. 10 % SDS solution: take 5g of SDS dissolve in 50 ml water.
3. 30 % acrylamide: 29.2g of acrylamide + 0.8g
methylenebisacrylamide in 100 ml water.
4. 1M NaH2PO4: take 11.9g dissolve and make the volume up to 100
ml with distilled water.
5. 0.5 M Na2HPO4: take 7.089g dissolve and make the volume up to
100 ml with distilled water.
6. Bromophenol blue 0.25 % w/v: take 25mg of bromophenol blue
and make up to 10 ml with distilled water.
7. TEMED (ready solution)
8. 2- mercaptoethanol (ready solution)
9. Glycerol (ready solution)
10. Reservoir buffer: mix 50 ml of tris/glycine with 5 ml SDS, make up
the volume to 500 ml with distilled water.
11. Staining solution:
1.25 g coommassie blue
230 ml methanol
230 ml water
40 ml acetic acid
12. Destaining solution:
Same as staining solution but with out the coommasie blue
Methanol
Water
Acetic acid
Gel preparation solution:
10 ml
1 ml
33.3 ml
0.15 ml
54.8 ml
99.25ml
1M tris- 1M glycine
10% SDS
30% acrylamide
TEMED
H2O
gel initiated by the addition of 0.75 ml of freshly prepared 10% ammonium
per sulphate ( 1 g in 10 ml water )
Sample preparation buffer:
4 ml
8 ml
4 ml
1.7 ml
0.5 ml
1.0 ml
glycerol
SDS 10%
2- mercaptoethanol
1 M NaH2PO4
0.5 M Na2HPO4
bromophenol blue
20 µl of the sample preparation buffer is mixed with 100 µl of sample prior to
heating (heat for 2 minutes)