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Protocols from Sales et al. (2020) Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and 14C-based assays

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Abstract

This collection of protocols includes four assays for measuring Rubisco activity that are used and discussed in the manuscript "Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and 14C-based assays" (Sales et al. 2020).

Three of the protocols are NADH-linked microtiter plate-based assays for Rubisco activity that use alternative coupling enzymes:
1. GAPDH-GlyPDH, glyceraldehyde-3-phosphate-dehydrogenase and glycerolphosphate-dehydrogenase;
2. PEPC-MDH, phosphoenolpyruvate-carboxylase and malate-dehydrogenase;
3. PK-LDH, pyruvate-kinase and lactate-dehydrogenase.

The radiometric assay for Rubisco activity measures the incorporation of 14CO2 into 3-phosphoglycerate (3-PGA).

The fifth protocol describes the purification of 2,3-bisphosphate-dependent phosphoglycerate mutase (dPGM), which is required for the NADH-linked microtiter plate-based assays 2 (PEPC-MDH) and 3 (PK-LDH).