GAPDH | Glyceraldehyde-3-phosphate dehydrogenase (chloroplastic)

Variable amounts of purified spinach AB-GAPDH enzyme (0.32, 0.8, 2, and 5 µg/well) and protein extract (8, 20, 50 and 125 µg/well) from Spinacia oleracea leaves were separated on 12% SDS-PAGE and blotted to a nitrocellulose membrane (0.2 µm) using the TransBlot (BioRad). Membrane was stained with Red-Ponceau and then blocked with TBST containing 5% skim milk for 1 h at RT. After blocking, the membrane was incubated at 4°C/ON in the presence of Agrisera primary antibody (1:1 000 dilution) in TBST containing 1% milk under continuous agitation. After incubation, the membrane was washed 5 times for 10 min in TBST with 1% milk at RT and then incubated with Agrisera secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) at 1:25 000 dilution for 90 min at RT. The membrane was then washed 5 times for 10 min in TBST with 1% milk and TBST for an additional 20 min prior to incubation (2 min) with Agrisera ECLBright (AS16 ECL-N). The signal was acquired using the LAS500 instrument (Cytiva) after exposure times of 30 and 80 sec. ​ ​
The GAPDH enzyme was purified according to Fermani et al. (2007)

Courtesy of Dr. Aitziber Calleja, University of Navarra, Spain