Bitan lab wiki. Estimation of methionien sulfoxide reductase (Msr) activity by HPLC method

Estimation of methionine sulfoxide reductase (Msr) activity by HPLC method

Total Msr activity was measured from rat primary cortical neurons by HPLC method

Reagents and accessories:

DMEM, fetal bovine serum, streptomycin/penicillin (Invitorgen), paraformaldehyde, Poly D-lysine, cytosine β-D arabinoside, 60 mm petriplate, dabsyl-Met-sulfoxide, DTT, Tris, acetonitrile (Sigma).

Protocol:

The cortical neurons were prepared from E18 embryos and culture for 6 days in vitro (DIV).

The neurons were treated with peptides (final concentration 10 μM, pretreated with 60 mM NaOH and dissolve in DMEM media) for 24 hours.

After treatment the media was removed and wash for three times with PBS.

Then the cells were lysed by sonication under ice-cold condition, spun-down the pellets and protein content was measured from supernatant Then from supernatant, the Msr activity was measure by the following way

Dabsyl-Met sulfoxide [Dabsyl-Met (O)] was used as substrate for Msr activity. The reaction mixture contains: 20 mM DTT, 50 mM Tris 7.5 and 200 μM Dabsyl-Met(O). Then it was incubated for 1 hr at 37oC and the reaction was stopped with equal volume of acetonitrile and injected to C-18 column for analysis the soluble material (absorbance: 435nM) with gradient from 0.14M sodium acetate pH 6.0 to 70% acetonitrile for up to 30 min).

The result was normalized with control and expressed as percentage changes of Msr activity.