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Iso-enzyme analysis
Iso-enzyme analysis was done from the shoot and inflorescence apex at different life
phase from both the Normal and Mutant Beta palonga.
i.
Extraction of enzymes:
Enzyme was extracted following the method of Wetter and Dyck (1993). Two
grams of sample (shoot and inflorescence apex) were frozen first in liquid
nitrogen and then crushed with mortar and pestle and 5 ml ice cold (4C)
extraction buffer was added (Table: 3). The macerates were centrifuged at
12,000 rpm for 20 minutes at 4C to collect the supernatant containing the
extracted enzymes. Finally the sample was stored at -20C.
Composition
Amount
Storing condition
Plant iso-enzyme
extraction buffer
Tris-base
Sucrose
2-marcaptoethanol
H2O upto
2.422 gm
34.230 gm
0.4 ml
100 ml
4 0C
pH 8.5
ii.
Extraction of enzymes:
on gel surface and then the comb was placed over the gel surface
and finally the gel was allowed to stand for 2-3 hours.
b. Stacking gel: Measured amount of stock solutions were mixed for
stacking gels as mentioned in table-4. The mixture was degassed
prior to the addition of ammonium per sulphate and TEMED. The
running gel was poured off and stacking gel solution was gently
poured into the sandwich over polymerized separating gel. A comb
was inserted into the sandwich to make wells in the stacking gel at
regular interval for application of sample proteins. Finally the
stacking gel was allowed to polymerize for half an hour.
iv.
Composition
Amount
Storing condition
Tris-HCl
H2O upto
18.17 gm
100 ml
4 0C
Tris-HCl
H2O upto
3.02 gm
50 ml
Acrylamide
monomer
Bis-Acrylamide
H2O upto
29.20 gm
0.80 gm
100 ml
Solution-A
1.5 M Tris HCl
buffer
(pH adjusted with conc. HCl)
pH 8.8
Solution-B
0.5 M Tris HCl
buffer
(pH adjusted with conc. HCl)
4 0C
pH 6.8
Solution-C
30% Monomer
Acrylamide solution
40C
Solution-D
10% Ammonium
per-sulphate
solution
Ammonium
per-sulphate
H2O upto
0.10 gm
1 ml
Freshly prepared
Tris HCl
Glycine
H2O upto
3.02 gm
14.41 gm
1 liter
Room temperature
Running buffer
0.025 M Tris
0.192 M Glycine
v.
Solutions
Separating gel
(8.5%)
Solution-A
Solution-B
Solution-C
Solution-D
TEMED
H2O
4.00 ml
5.66 ml
0.1 ml
10 l
10.04 ml
0.50 ml
0.675 ml
50 l
5 l
3.75 ml
pH
Compositions
Amoun
t
0.2 M Phosphate
buffer
6.0
NaH2PO4, 2H2O
Na2HPO4
ddH2O upto
2.08 gm
0.94 gm
100 ml
0.2 M Phosphate
buffer
7.5
NaH2PO4, 2H2O
Na2HPO4
ddH2O upto
2.83 gm
0.30 gm
100 ml
Acetate-B buffer
5.0
2.06 ml
1.12 gm
200 ml
Tris-A buffer
8.0
Na-EDTA
Tris
ddH2O upto
pH adjust with conc.
HCl
80 mg
4.84 gm
200 ml
Tris
ddH2O upto
pH adjust with conc.
HCl
1.21 gm
100 ml
7.5
dark at 300C. Next day the blue bands developed which indicated
the activity of this enzyme (Wendel and Weeden, 1989).
Staining ingredients
Quantity
Acid Phosphatase
0.1 gm
0.1 gm
100 ml
GOT
SOD
PGD
MgCl2
82 ml
1 mg
100 mg
400 mg
64 mg
2.24 mg
136 mg
100 ml
Tris-A buffer
MgCl2 (0.5 M)
NAD (1%)
Before use add
NBT (0.1%)
PMS (1%)
40.00 ml
0.20 ml
1.00 ml
100 ml
98 mg
20 mg
15 mg
20 mg
4 mg
Phosphogluconic acid
NADP
MTT
PMS
1.00 ml
0.50 ml
6-