Anti-Tubulin alpha chain (polyclonal antibodies)

Western Blot

1 - 20 μg of Arabidopsis thaliana whole rosette leaves extract
2 - 10 μg of Arabidopsis thaliana whole rosette leaves extract
3 - 5 μg of Arabidopsis thaliana whole rosette leaves extract

20, 10 and 5 μg of total protein from Arabidopsis thaliana rosette leaves extracted with extraction buffer 100 mm Tris-HCl, pH 8.5, 4% [w/v] SDS, 2% [v/v] β-mercapthoethanol, and 2 mm phenylmethylsulfonyl fluoride and separated on 10% SDS-PAGE and blotted 1h to PVDF. Blots were blocked with 1% non-fat milk in TBS for 1 h at room temperature with agitation. Membranes were incubated in the primary antibody at a dilution 1:5000 (A) or 1:1000 (B) in 1% non-fat milk in TBS at 4°C overnight. Then, membranes were washed 3 Times for 15 min in TBS at RT with agitation. Membranes were incubated in secondary antibody (Anti-rabbit IgG horse radish peroxidase conjugated from Agrisera AS09 602) diluted to 1: 25 000 in 1% non-fat milk in TBS for 1h at RT with agitation. The blots were washed as above and developed for 5 min with ECL (Agrisera ECL Bright AS16 ECL-N) according to the manufacturer's instructions. Exposure time was 2 seconds.

Courtesy of Dr. Agnieszka Zienkiewicz, Centre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University, Toruń, Poland

 5 µg of total protein from Arabidopsis thaliana (1), Hordeum vulgare (2), Zea mays (3)   extracted with Agrisera PEB extraction buffer were separated on  4-12 % SDS-PAGE and blotted 1h to PVDF. Blots were blocked with   for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 10 000 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-rabbit IgG horse radish peroxidase conjugated, from Agrisera AS09 602 ) diluted to 1:25 000 in  for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL Advance according to the manufacturers instructions (GE Health care). Exposure time was 120 seconds.

Total protein from either rice embryos or rice protoplasts extracted with buffer containing 60mM Tris-HCl (pH8.0), 2% SDS(w/v), 15% Sucrose (w/v) and protease inhibitor 1X were separated on 12 % SDS-PAGE and blotted 1h to PVDF. Blots were blocked with skimmed milk containing TBS 1X for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 overnight at 4 degree with shaking about 40 rpm. 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-rabbit IgG horse radish peroxidase conjugated) diluted to 1:20 000 in for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturers instructions. Exposure time was 10 seconds.

Courtesy of Ho Viet The, PhD Student, Scuola Superiore Sant'Anna, Pisa, Italy

Immunolocalization

Tubulin alpha localization in roots of Arabidopsis thaliana.  Tubulin alpha (red), nucleus (DAPI white). Plant material has been fixed in para-formaldehyde for 30 minutes. Tissue cleaning has been performed before immunolocalization. Primary antibodies: Agrisera anti-tubulin alpha 1: 500. Secondary antibody:  goat anti-rabbit IgG Alexa conjugated (red color), dilution 1: 500. Scale bar – 10 µm.

Courtesy Dr. Taras Pasternak, Freiburg University, Germany

Tubulin alpha (TUA) together with beta tubulin is making up microtubules.