TKL1 | transketolase (chloroplastic)

application example

Approximately 1 ug (in dilution series) of total soluble (stromal) protein of Arabidopsis thaliana extracted from isolated chloroplasts using a buffer containing 10% glycerol, 200 mM NaCl, protease inhibitor and 1mM DTT. Samples were denatured at 96°C for 3 min and were separated on 10 % SDS-PAGE  and blotted for 45min. to PVDF using semi-dry transfer. Blots were blocked with TBST with 5 % milk powder (0.02 % Tween 20)  for 1h at room temperature (RT). Blot was incubated in the primary antibody at a dilution of 1: 5 000 for overnight at 4°C with agitation in TBST with 5 % milk powder. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 10 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 12:0 000 in  for 1h at RT with agitation. The blot was washed as above and developed following manufacture recommendations. Exposure time was 10-30 seconds.

Courtesy of Dr. Ute Vothknecht and Edoardo Cutolo, Munich University, Germany