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APPENDIX

1. 0.5M Tris-HCl (100ml)
• Tris: 6.05g
• Dissolve in 70ml distilled water
• Adjust the pH to 6.8 with HCl and make up to 100ml


• Store at 4°C
2. 1.5M Tris-HCl 100ml)
• 18.15g
• Dissolve in 70ml distilled water
• Adjust the pH to 8.8 with HCl and make up to 100ml
• Store at 4°C
3. 10% Amino persulphate (10% APS) (1ml)
• Ammonium persulphate: 100mg
• Dissolve in 70ml distilled water (prepare fresh everytime)
4. 10% resolving (2gel)
• 30% Bis-Acrylamide: 6.075ml
• 1.5M Tris-HCl (pH=8.8): 4.95ml
• 10% SDS: 150µl
• DI water: 3.75ml
• TEMED: 7.5 µl
• 10% APS: 75 µl
5. 10% SDS (100ml)
• SDS: 10g


O : 90ml
41


6. 12% resolving
• 30% Bis-Acrylamide: 5.94ml
• 1.5M Tris-HCl (pH=8.8): 3.75ml
• 10% SDS: 150µl
• DI water: 5.085ml
• TEMED: 7.5 µl
• 10% APS: 75 µl
7. 4% stocking
• 30% Bis-Acrylamide: 0.99ml
• 1.5M Tris-HCl (pH=8.8): 1.89ml
• 10% SDS: 75µl
• DI water: 4.5ml
• TEMED: 7.5 µl
• 10% APS: 37.5 µl
8. Binding buffer (1L)
Make 20mM sodium phosphate
• 0.2M di-sodium phosphate (

.2

) (100ml)

di-sodium phosphate : 3.56g
Dissolve in 100ml distilled water
• 0.2M

.

O (100ml)

mono-sodium phosphate : 2.76g
Dissolve in 100ml distilled water
• Adjust pH by mixing mono-sodium phosphate and di-sodium phosphate
• Mix 100ml Sodium phosphate and 900ml distilled water

42


9. Blocking buffer 3% BSA (50ml)
• BSA: 1.5g
• Add 30ml PBST
• Mix well up to 50ml
10. Blocking buffer 5% skim milk (50 ml)
• Skim milk: 2.5g
• Add PBST and mix well up to 50ml
11. Destanining Solution (500ml)
• Methanol: 200ml
• Acetic acid: 35ml
• Up to 500ml with distilled water
12. Developeing buffer (5ml) (AP conjuagate substrate kit)
• Development buffer: 200ml
• Reagent A: 50µl
• Reagent B: 50µl
• DI water: 4.7ml
13. Developing solution (100ml)
• 500µl of 1% citric (0.01g/1ml DI)
• 50µl 37% formaldehyde
• Up to 100ml with DI water
14. Elution buffer (1L)
Make 20mM sodium phosphate
• 0.2M di-sodium phosphate (

.2

di-sodium phosphate : 3.56g
Dissolve in 100ml distilled water
43

) (100ml)


• 0.2M

.

O (100ml)

mono-sodium phosphate : 2.76g
Dissolve in 100ml distilled water
• Adjust pH by mixing mono-sodium phosphate and di-sodium phosphate
• Mix 100ml Sodium phosphate and 900ml distilled water
Mix 100ml Sodium phosphate with 2M NaCl and up to 1L with distilled water
15. PBS 10X (250ml, pH=7.4)
• NaCl: 20g
• KCl: 0.5g


: 0.6g



: 3.6g

• Dissolve in 100ml distilled water and adjust pH by NaOH up to 7.4
16. PBST (500ml)
• 1X PBS: 497.5ml
• 0.5% Tween 20: 2.5ml
17. Running buffer 10X (500ml, pH=8.3)
• 250mM Tris: 15.15g
• 1.92M glycerin: 72g
• 1% SDS: 2.5g
• Dissolve in distilled water and up to 500ml
18. Silver nitrate solution
• 0.8g Ag

/ 4ml DI water

• 0.36% NaOH: 21ml


OH: 1.4ml

• Up to 100ml with DI water
44


19. Staining solution (500ml)
• Commassie blue: 0.125g
• Methanol: 200ml
• Acetic acid: 35ml
• Adjust volume by distilled water
20. Transfer buffer (500ml)
• Methanol: 100ml
• Tris base: 7.21g
• Glycine: 1.52g
• Up to 500ml with distilled water

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