Enzyme Kinetics assay of the WT
To assay 17 b-HSD activity in lysates, cells were harvested 48h after transfection using PBS Enzyme Free Cell Dissociation Solution ( specialty Media Inc., Lavellette, NJ), frozen on dry ice and stored at -80 C.
Cell pellets were thawed on ice, resuspended in :
* 10mM Tris-HCl(pH 7.4)/150mM KCL/1 mM EDTA/2mM Dithiothreitol (DTT) at protein concentration of 3-6 mg/ml, and snap-frozen on dry ice . After thawing at 37° C, the extracts were chilled on ice and sonicated for 30sec.
A typical assay for establish the apparent Vmax and Km of the Substrate ANDROST-4-ENE-3,17-DIONE of the normal & mutant HSD17B3 enzyme used:
* 20-30 microgram of total protein in 0.2 mL of 100mM Tris - citrate(pH6)/2mM DTT.
* Steroid substrate was added in different concent.(0.1-8 microM), and NADPH was added to a final cocent. of 2mM.
* Reactions were initiated by the addition of enzyme and were carried out for 25’- 30’ min at 37 ° C .
A typical assay to establish the apparent pH of the normal & mutant HSD17B3 enzyme used:
* Serial variation of pH (range4.5pH -8.5pH), for the reaction Buffer : 100mM Tris -citrate/2mM DTT.
* Steroid substrate was added at final concent. of 5 mM ( where 1 mM were 14C- ANDROST-4-ENE-3,17-DIONE and 4mM were cold substrate).
Transfection of DNA into 293T cells by using LIPOFECTAMINE PLUS Reagent package (LIFE TECHNOLOGIES Cat.No.10964-013):
Protocol
* The day before transfection, split and plating the cells, so the that they are 50%- 60% confluent the day of transfection. Avoid antibiotics at the time of transfection and during.
* Culture vessel:100mm
* plasmid DNA ; 4 microg
* plasmid b-gal ; 1 microg
* PLUS reagent; 20 microliter
* LIPOFECTAMINE PLUS Reagent; 30 microliter
* Incubate at 37°C at 5% CO2 for 3h (see LIFE TECHNOLOGIES protocol.)
* After 3h inc., increase volume of medium to normal volume (see LIFE TECHNOLOGIES protocol.)
* The cell extract were assayed for reporter gene activity 48h after the start of transfection.
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2008-2-3