PK/LDH Coupled Assay.

Pyruvate kinase/lactate dehydrogenase coupled assay 

Overview:

This is a coupled enzyme assay that indirectly measures ATP consumption by coupling the ADP produced to NADH oxidation via:

• Pyruvate Kinase (PK): converts ADP + PEP → Pyruvate + ATP
• Lactate Dehydrogenase (LDH): converts Pyruvate + NADH → Lactate + NAD⁺

The decrease in NADH absorbance (typically at 340 nm) or fluorescence (excitation ~340 nm, emission ~460 nm) reflects ATP consumption by the kinase of interest (e.g., Btk).

Stock Solutions Needed:

• PK/LDH enzyme solution (Sigma)
• 1M HEPES, pH 7.4
• 5M NaCl
• 50% glycerol
• 0.1M ATP, pH 7.0
• 500mM PEP
• 1M DTT
• 2M MgCl₂
• ~40mM NADH (verify via A₃₄₀ using ε = 6220 M⁻¹cm⁻¹)

Note: NADH solution is prone to degradation via oxidation. Aliquot and store at –80°C to minimize oxidation.

Reaction Buffer Recipes (Per 10 mL):

NADH Standards:

Prepare using No NADH/ATP buffer and 40mM NADH stock:

• 0 μM
• 50 μM
• 100 μM
• 200 μM
• 400 μM
• 600 μM (used for auto-gain in plate reader)

Assay Setup (50 μL Reactions):

1. Thaw buffers as needed (2x NADH/ATP buffer should be thawed later, just before it is added to the reactions.)
2. Place a Black/Clear bottom 96-well microplate on ice.
3. Prepare enzyme/buffer mix:
   Mix the PK/LDH solution with the No NADH/ATP buffer in a 1:13 ratio (this ratio can be adjusted accordingly).
4. Plate Setup:
   • Add 20 μL enzyme/buffer mix to each BLANK well (no kinase) and each ASSAY well (with kinase).
   • Add 5 μL SEC buffer to BLANK wells.
   • Add 5 μL of 20 μM Btk stock to ASSAY wells (final = 2 μM for FL Btk assays).
5. Add 50 μL of each NADH standard to the wells of the second-to-last row.
6. Thaw 2x NADH/ATP buffer 
7. Transfer 2x NADH/ATP buffer to the wells of the bottom row, allowing a slight excess over the volume needed (~30 μL per reaction) so the multi-channel pipette does not draw air
8. Bring the following to the plate reader: the microplate (kept on ice), a multi-channel pipette, pipette tips, and a syringe needle.

Running the Experiment:

1. Prepare the plate reader (set protocol, lamp warm-up).
2. With a multi-channel pipette, add 25 μL 2x NADH/ATP buffer into each reaction (BLANK & ASSAY) to initiate.
3. Remove air bubbles using a needle to avoid errors in reading.
4. Immediately begin reading at 460 nm (NADH emission).