Anti-tdTomato

Samples:

1 - 20 ug of Arabidopsis thaliana transgenic line expressing Lti6b-tdTomato 7d old control sample (whole seedlings)
2 - 20 ug of Arabidopsis thaliana 4d old of control sample (root) Mark: MW markers BlueStar prestained protein marker (Nippon Genetics)

20 µg/well of total protein extracted freshly from Arabidopsis thaliana 7d old seedlings. Exact buffer components were: 25 mM Tris-HCl pH 7,6, 150 mM NaCl, 1% v/v NP-40, 1% w/v sodium deoxycholate, 0,1% w/v SDS and Complete™ Protease Inhibitor Cocktail (Roche) and denatured with 4x Laemnli buffer (1x: 50 mM Tris-HCl pH 6.8, 2% w/v SDS, 0.1% w/v bromophenol blue, 10% v/v glycerol and 2.5% v/v β-mercaptoethanol) at 85 °C/10 min and. Samples were separated on 12% SDS-PAGE and blotted for 60 min to PVDF (pore size: 0.45 μm), using: wet transfer at 4°C. Blot was blocked with 5 % non-fat milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:500 with agitation in TBS-T,  ON/4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly thrice for 30 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 25 000 for 2h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: AgriseraBright (Agrisera). Exposure time was 30 sec.

Note: Protein load/well can be decreased and primary antibody used in higher dilution, which will contribute to less background. 

Courtesy of phd candidate Veronica Giourieva, Department of Botany School of Biology, Aristotle University of Thessaloniki 54124 Thessaloniki, Greece