2,4-dinitrophenylhydrazine alpha-ketoglutarate detection assay for Prolyl Hydroxylase Domain (PHD) proteins

Prepare 5 Eppendorf tubes containing 50 µl of 10% TCA.

• Label tubes: 0 min, 1 min, 2 min, 5 min, 15 min.

Prepare cofactor solution containing HEPES/MES, catalase, DTT, ascorbic acid, FeSO₄, a-ketoglutarate, and peptide in a 150 µl volume in an Eppendorf tube (using the working concentrations).

Add 150 µl of 20 µM PHD enzyme into the cofactor solution.

Vortex briefly.

Place into a 37 ^oC tabletop shaking incubator and start the timer (counting up).

• This step equilibrates the temperature of the reaction to 37 ^oC

At T = 1 min on the timer, withdraw 50 µl of the reaction solution and quench in the “0 min” tube containing 10% TCA, and replace the reaction tube in the incubator.

Repeat this for the other time points.

• At T = 2 min, withdraw 50 µl of the reaction solution and quench in the “1 min” tube
• At T = 3 min, withdraw 50 µl of the reaction solution and quench in the “2 min” tube
• At T = 6 min, withdraw 50 µl of the reaction solution and quench in the “5 min” tube
• At T = 16 min, withdraw 50 µl of the reaction solution and quench in the “15 min” tube

Briefly vortex the quenched reactions.

Keep the quenched reactions at 4 ^oC until ready for downstream processing.

• Reactions have been stored up to 3 days with no loss of signal.

Centrifuge the quenched reactions at 13,000 rpm for 15 minutes.

Meanwhile, add 10 µl of 0.5 M sodium phosphate to 5 wells of a 96-well plate.

Transfer 90 µl of the supernatant of the quenched reaction to a well containing 10 µl of 0.5 M sodium phosphate (V_T = 100 µl).

• Do the same for the other 4 quenched supernatants.

Using a multi-channel pipette, add 100 µl of 50 mM 2,4-DNPH to the wells (V_T = 200 µl). Pipette up and down gently to mix.

Leave at room temperature for 20 minutes.

Using a multi-channel pipette, add 50 µl of 6 M NaOH to the wells (V_T = 250 µl). Pipette up and down gently to mix.

Leave at room temperature for 5 minutes.

Read at 425 nm on a spectrophotometer.

Calculate the amount of a-ketoglutarate consumed from a standard curve processed in the same way as the samples.

Plot the amount of a-ketoglutarate consumed against time, to obtain a curve that looks like this:

The initial rate should be taken as the linear portion of the curve. In this case, from T = 0 to 2 mins.