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Appendix 1
Phosphate buffered saline
NaCl
- 8 gm
KCl
- 0.2 gm
Na2HPO4
- 2.88 gm
KH2PO4
- 0.2 gm
pH
- 7.4
All the ingredients are weighed and dissolved in 1000 ml of sterile Millipore water. The
solution was filtered through Whatman filter paper No 4 and autoclaved at 15 lbs for 15
mins.
Appendix 2
Trypsin Versine Glucose
Phosphate buffered saline
- 840 ml
2% Trypsin
- 50 ml
0.2 % EDTA
-100ml
10% Glucose
- 5ml
Phenol red
- 5ml
All the ingredients are mixed and the pH was adjusted to 7.4 by 0.1N HCl or 0.1 N NaOH
and stored at -20˚C.
Appendix 3
Separating Gel Mixture
Acrylamide
- 3.3ml
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Tris buffer (pH 8.8)
- 2.5ml
SDS (10%)
- 100µl
APS (10%)
- 100µl
TEMED
-10µl
Distilled water
-3 ml
Appendix 4
Stacking Gel Mixture
Acrylamide
- 660 µl
Tris buffer (pH 8.8)
- 1.25 µl
SDS (10%)
-50 µl
APS (10%)
- 50µl
TEMED
-10µl
Distilled water
-1.5 ml
Appendix 5
Cell Lysis buffer
1M Tris HCl 7.4
- 2.5ml
1M NaCl
-1.5 ml
Triton X
- 500 µl
EGTA 0.5 M
-200 µl
100 mM PMSF
-1/100 of total volume of buffer
These are added
Leupeptin(1 µg/ml)
-1/100 of total volume of buffer
just before use
Aprotinin (1 µg/ml)
-1/100 of total volume of buffer
Appendix 6
Phosphate buffer (pH 8.0)
KH2PO4 (0.2 M) – 1.3609 gm dissolved in 50 ml water Solution A
NaOH (0.2 M) - 0.4gm dissolved in 50 ml water Solution B
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50 ml of solution A and 46.1 ml of solution B are mixed and the final volume was made up
with 100 ml with distilled water
Appendix 7
DTNB (Dithiobis 2-nitrobenzoic acid) 34 mg of DTNB is weighed and mixed with 10ml of
1 % trisodium citrate solution
CDNB (1 chloro 2,4 dinitrobenzene) 40.5 mg of CDNB in 10 ml of absolute ethanol.