Use fresh 10 x 75 mm tubes, place on ice
Place x lambda of sample in tube (for column fractions use 1ul, cells
20ul); include a "no sample" of just buffer for a zero (100ul)
Add 100-x ul of 50mM NaOAc pH 5.1, /- 0.1% TX-100
Start reaction by adding 25 ul of 0.5M sucrose (keep sucrose stored at -20
in 1 ml aliquots), incubate tubes at 37oC for 3-20 min (varies dep. on
amount of invertase expected, for column 5 min)
Add 150 ul 0.2M K2HPO4, place tubes on ice stops reaction
Boil 3 min, then place tubes on ice, now assay liberated glucose
Add 1 ml of assay mix:
50 ml 0.1M KPi buffer, pH 7.0
100 ul glucose oxidase 5000 U/ml (in 4M
NaCl)
125 ul peroxidase 1 mg/ml
750 ul O-dianisidine 10 mg/ml
Incubate at 37oC for 30 min
Add 1 ml of 6N HCL
Read A540 against the zero
STANDARD CURVE
use 1 mg/ml glucose in water
make following samples:
amount of 1mg/ml glucose amount of 50mM NaOAC/0.1% TX-100
0 ul 0 ug 125 ul
2 2 123
5 5 120
10 10 115
15 15 110
20 20 105
Add 150 ul K2HPO4
Add 1 ml assay mix
Incubate at 37oC for 30 min
Add 1 ml 6N HCL
Read A540
For external and internal invertase assays on cells do the following:
For 1 ml cell culture in 10 mM NaN3:
Total:
Remove 0.5 ml cells in NaN3
Add 0.5 ml of 2X spheroplast cocktail mix: FOR 5 ml
2.8 M sorbitol, 0.1M Tris pH 7.5
10mM NaN3
20 ul beta-ME
0.5 mg Zymolyase-100T
Incubate 45 min at 37oC AVOID LYSIS
Pellet 5 min in clinical, remove sup. with drawn out pipet
Lyse in 0.5 ml 0.5% TX-100
Assay 20 ul in 80 ul of 0.1M NaOAc, pH 5.1 in above assay
NOTE: Include NEM (100ul of a 10mM stock per 50 ml of assay mix) to
neutralize beta-ME
Internal
Assay 20 ul of cell culture directly in 80 ul 0.1M NaOAc pH 5.1
NO TX-100
Perform above assay
before reading tubes, spin 2 min in clinical
Internal= Total - above reading
remember no sample, tubes of 20 ul NaN3 or 20 ul 0.5% TX-100
Read A600 0f 1:10 dilution of whole cells in NaN3 so can normalize to OD
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