STUDY OF SERUM ELECTROPHORESIS
Introduction:
Electrophoresis is an analytical technique for the separation of charged compounds and purify the molecules. e.g. Proteins, lipoproteins, enzymes and nucleic acids.
It is used as diagnostic tool in medical field for diseases like multiple myeloma, kala-azar, etc.
Principle:
It is an analytical method for separation of charged particles in an electric field resulting in their migration towards the oppositely charged electrode.
Molecules with a net positive charge (cations) move towards the negatively charged electrode (cathode) while that with a net negative charge (anions) move towards the positively charged electrode (anode).
The velocity of migration of each molecule in an electric field is dependent upon the net charge on the molecule, strength of the electric field, the medium and is inversely proportional to the molecular weight.
Materials Required
Apparatus: Electrophoresis tank, Power unit, Slides, Glass plate, Wicks, Spikes
Reagents:
1. Buffer(Barbitone & Sodium barbitone)
pH=8.6
Barbitone(0.92gm)+ Sod. Barbitone(5.15gm) to 500mL D/W
2. Agarose(1%,0.1 gm in 10mL buffer)
3. Amido black(0.5% in 5% Acetic Acid)
4. Control serum
5. Test serum
6. Bromophenol blue(as Tracking Dye)
7. Methanol
8. Acetone
9. 3 % Acetic acid
Procedure:
1. A clean glass plate is taken and at two places a control serum and a test serum is applied with a spike in each and bromophenol blue as a tracking dye
2. Mix two of them separately
3. A slide is prepared by pouring over melted agar on it uniformly
4. Then spikes loaded with the control and test serum is appied on the slide in two rows towards the left edge, which will be a cathode end.
5. The two compartment in electrophoresis tank is filled with chilled buffer in equal volume(about 100mL)
6. Then, the slide is kept in between the tanks and wicks(wetted with buffer) are applied to complete the circuit
7. Then current of 5-7 mAmp per slide is applied and left for 2-3 hours until the tracking dye reaches the anode end of slide
8. Then it is taken out and fixed in methanol for 10 min
9. It is then dehydrated in acetone for 5 min and kept overnight for drying or at 700C in an oven for an hour.
10. Then the slide is stained with 0.5% Amido black
11. The slide is washed with 3% acetic acid
OBSERVATION
A dark albumin band is seen at right hand side of slide and α1, α2, β and γ-bands towards its left respectively.
RESULT and DISCUSSION
Electrophoresis of the serum resulted in the separation of its proteins(both control and test).
The γ-band of the test was found to be increased, which is the case in diagnosing multiple myeloma.
CONCLUSION
By the process of electrophoresis the test serum was diagnosed to be of multiple myeloma.
PRECAUTION
1. Spikes should be of equal in size for both test and control to load equal amount
2. Agar is to be spread in slide uniformly and as fast as possible since it cools down quickly
3. Wicks should be applied correctly, and checked during procedure
4. Drying of the slide is better done for overnight
REFERENCES
1. Rajagopal G., Toora B.D., 2005, Practical Biochemistry, 2nd edition, Pg. no. 177-185
2. Chatterjea M.N., Shinde R.,2007, Textbook of Medical Biochemistry, 7th edition, Pg. no. 772-775
Introduction:
Electrophoresis is an analytical technique for the separation of charged compounds and purify the molecules. e.g. Proteins, lipoproteins, enzymes and nucleic acids.
It is used as diagnostic tool in medical field for diseases like multiple myeloma, kala-azar, etc.
Principle:
It is an analytical method for separation of charged particles in an electric field resulting in their migration towards the oppositely charged electrode.
Molecules with a net positive charge (cations) move towards the negatively charged electrode (cathode) while that with a net negative charge (anions) move towards the positively charged electrode (anode).
The velocity of migration of each molecule in an electric field is dependent upon the net charge on the molecule, strength of the electric field, the medium and is inversely proportional to the molecular weight.
Apparatus: Electrophoresis tank, Power unit, Slides, Glass plate, Wicks, Spikes
Reagents:
1. Buffer(Barbitone & Sodium barbitone)
pH=8.6
Barbitone(0.92gm)+ Sod. Barbitone(5.15gm) to 500mL D/W
2. Agarose(1%,0.1 gm in 10mL buffer)
3. Amido black(0.5% in 5% Acetic Acid)
4. Control serum
5. Test serum
6. Bromophenol blue(as Tracking Dye)
7. Methanol
8. Acetone
9. 3 % Acetic acid
Procedure:
1. A clean glass plate is taken and at two places a control serum and a test serum is applied with a spike in each and bromophenol blue as a tracking dye
2. Mix two of them separately
3. A slide is prepared by pouring over melted agar on it uniformly
4. Then spikes loaded with the control and test serum is appied on the slide in two rows towards the left edge, which will be a cathode end.
5. The two compartment in electrophoresis tank is filled with chilled buffer in equal volume(about 100mL)
6. Then, the slide is kept in between the tanks and wicks(wetted with buffer) are applied to complete the circuit
7. Then current of 5-7 mAmp per slide is applied and left for 2-3 hours until the tracking dye reaches the anode end of slide
8. Then it is taken out and fixed in methanol for 10 min
9. It is then dehydrated in acetone for 5 min and kept overnight for drying or at 700C in an oven for an hour.
10. Then the slide is stained with 0.5% Amido black
11. The slide is washed with 3% acetic acid
OBSERVATION
A dark albumin band is seen at right hand side of slide and α1, α2, β and γ-bands towards its left respectively.
RESULT and DISCUSSION
Electrophoresis of the serum resulted in the separation of its proteins(both control and test).
The γ-band of the test was found to be increased, which is the case in diagnosing multiple myeloma.
CONCLUSION
By the process of electrophoresis the test serum was diagnosed to be of multiple myeloma.
PRECAUTION
1. Spikes should be of equal in size for both test and control to load equal amount
2. Agar is to be spread in slide uniformly and as fast as possible since it cools down quickly
3. Wicks should be applied correctly, and checked during procedure
4. Drying of the slide is better done for overnight
REFERENCES
1. Rajagopal G., Toora B.D., 2005, Practical Biochemistry, 2nd edition, Pg. no. 177-185
2. Chatterjea M.N., Shinde R.,2007, Textbook of Medical Biochemistry, 7th edition, Pg. no. 772-775
