Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis
Stock Reagents and chemicals preparation:
S.
Reagent / chemical Stock Preparation
No
1 1.5 M Tris-HCl (pH – 6.8) 11.82 g in 50 mL of dH2O and adjusted the pH to 6.8.
2 1.5 M Tris-HCl (pH – 8.8) 11.82 g in 50 mL of dH2O and adjusted the pH to 6.8.
3 1 M Tris -HCl (pH-6.8) 3.152 g in 20 mL H2O
4 10% SDS 5 g in 50 mL of dH2O
5 10% APS 0.1 g in 1mL of dU2O
6 Bromophenol blue 0.3% 30 mg in 10 mL
Sample buffer 4X: store at 4ºC for 1-2 weeks
S. Reagent / chemical Working solution
No (10 mL)
1 1 M Tris -HCl (pH-6.8) 2.5 mL
2 SDS 1g
3 Bromophenol blue 0.3% 0.5 mL
4 Glycerol 4 mL
Make upto 10 mL and aliquot into 1mL for use
5 2-Mercapto ethanol (add into the aliquot) 50 uL
Sample preparation:
1. Inoculate the colonies into 10 mL of YNB ura- medium containing 2% galactose.
2. Incubate at 30 ºC for overnight.
3. Centrifuge the culture at 5000g for 5 minutes at room temperature.
4. Resuspend the pellets using 20 μL of dH2O.
5. Transfer into 2 mL microcentrifuge tubes.
6. Add 40 µL of sample buffer (becomes 2X).
7. Vortex for 10s.
8. Heat at 95º C for 5 minutes.
9. Store at -20 ºC until use.
Protocol:
1. Clean the plates and set it on the casting tray and check for the leakage using water.
2. Prepare resolving gel and pour it for three fourth of the plates.
, Gel preparation:
S. Reagent / chemical Resolving gel (12.5 % ) Stacking gel (4 %)
No
1 dH2O 3.174 mL 1.502 mL
2 30% Acrylamide – Bis acrylamide 4.166 mL 0.330 mL
3 1.5 M Tris-HCl (pH -8.8) 2.5 mL …………….
4 1.5 M Tris-HCl (pH – 6.8) …………… 0.63 mL
5 10 % SDS 100 µL 25 μL
6 10% APS 50 µL 12.5 µL
7 TEMED (add finally) 10 µL 1.25 µL
3. Pour dH2O or isopropanol to remove the bubbles and leave it for polymerising.
4. Pour off the water and take out the remining layer of water using tissue.
5. Prepare stacking gel and pour it on the resolving gel.
6. Place the comb and leave it for polymerising.
7. Place the plates into the running module and keep it inside the tank.
8. Add running buffer.
Running buffer preparation: (pH – 8.3)
S. No Reagent / chemical 1 litre
1 25 mM Tris base 3g
2 Glycine 14 g
3 SDS 1g
Add 500 mL of dH2O, dissolve and adjusted the pH to 8.3. Then make upto 1litre
9. Load 10 μL of samples onto the wells.
10. Add 2 μL of protein prestained ladder in a lane.
11. Set the voltage at 80V until the sample migrates upto resolving gel.
12. Increase the voltage to 120V .
1. Take out the gel carefully.
2. Cut the stacking gel and mark one of the ends.
3. Wash the gel in dH2O.
4. Keep the gel in Coomassie brilliant blue staining solution.
Stock Reagents and chemicals preparation:
S.
Reagent / chemical Stock Preparation
No
1 1.5 M Tris-HCl (pH – 6.8) 11.82 g in 50 mL of dH2O and adjusted the pH to 6.8.
2 1.5 M Tris-HCl (pH – 8.8) 11.82 g in 50 mL of dH2O and adjusted the pH to 6.8.
3 1 M Tris -HCl (pH-6.8) 3.152 g in 20 mL H2O
4 10% SDS 5 g in 50 mL of dH2O
5 10% APS 0.1 g in 1mL of dU2O
6 Bromophenol blue 0.3% 30 mg in 10 mL
Sample buffer 4X: store at 4ºC for 1-2 weeks
S. Reagent / chemical Working solution
No (10 mL)
1 1 M Tris -HCl (pH-6.8) 2.5 mL
2 SDS 1g
3 Bromophenol blue 0.3% 0.5 mL
4 Glycerol 4 mL
Make upto 10 mL and aliquot into 1mL for use
5 2-Mercapto ethanol (add into the aliquot) 50 uL
Sample preparation:
1. Inoculate the colonies into 10 mL of YNB ura- medium containing 2% galactose.
2. Incubate at 30 ºC for overnight.
3. Centrifuge the culture at 5000g for 5 minutes at room temperature.
4. Resuspend the pellets using 20 μL of dH2O.
5. Transfer into 2 mL microcentrifuge tubes.
6. Add 40 µL of sample buffer (becomes 2X).
7. Vortex for 10s.
8. Heat at 95º C for 5 minutes.
9. Store at -20 ºC until use.
Protocol:
1. Clean the plates and set it on the casting tray and check for the leakage using water.
2. Prepare resolving gel and pour it for three fourth of the plates.
, Gel preparation:
S. Reagent / chemical Resolving gel (12.5 % ) Stacking gel (4 %)
No
1 dH2O 3.174 mL 1.502 mL
2 30% Acrylamide – Bis acrylamide 4.166 mL 0.330 mL
3 1.5 M Tris-HCl (pH -8.8) 2.5 mL …………….
4 1.5 M Tris-HCl (pH – 6.8) …………… 0.63 mL
5 10 % SDS 100 µL 25 μL
6 10% APS 50 µL 12.5 µL
7 TEMED (add finally) 10 µL 1.25 µL
3. Pour dH2O or isopropanol to remove the bubbles and leave it for polymerising.
4. Pour off the water and take out the remining layer of water using tissue.
5. Prepare stacking gel and pour it on the resolving gel.
6. Place the comb and leave it for polymerising.
7. Place the plates into the running module and keep it inside the tank.
8. Add running buffer.
Running buffer preparation: (pH – 8.3)
S. No Reagent / chemical 1 litre
1 25 mM Tris base 3g
2 Glycine 14 g
3 SDS 1g
Add 500 mL of dH2O, dissolve and adjusted the pH to 8.3. Then make upto 1litre
9. Load 10 μL of samples onto the wells.
10. Add 2 μL of protein prestained ladder in a lane.
11. Set the voltage at 80V until the sample migrates upto resolving gel.
12. Increase the voltage to 120V .
1. Take out the gel carefully.
2. Cut the stacking gel and mark one of the ends.
3. Wash the gel in dH2O.
4. Keep the gel in Coomassie brilliant blue staining solution.
