Anti-DYKDDDDK (binds to Sigma FLAG®, clone FG4R)

20 µg of total protein from Chlamydomonas reinhardtii   Flag; 2  (1) 1xFlag 5’+ICL (1x FLAG coding sequence : GA-CTA-CAA-GGA-CGA-CGA-TGA-CAA-G) (2); 1xFlag 5’+ICL (1x FLAG codingsequence :GA-CTA-CAA-GGA-CGA-CGA-TGA-CAA-G) (3); ICL+ 3xFlag 3’end (3xFLAG codingsequence : GAC TAC AAG GAC CAC GAC GGT GAC TAC AAG GAC CAC GAC ATC GAC TAC AAG GAC GACGACGAC AAG TGA.) (4); 1xFlag 5’+ICL+ 3xFlag 3’end (5) were separated on 12 % SDS-PAGE  and blotted ON (30V) to PVDF using tank transfer. Blots were blocked with 5 % non-fat milk  for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 5 000 for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-mouse IgG horse radish peroxidase conjugated) diluted to 1:10 000 in  for 1h at RT with agitation. The blot was washed as above and developed for 5 min with BM ECL kit according to the manufacturer's instructions. Exposure time was  seconds.