Amborella trichopoda, Brassica napus, Capsella rubella, Citrus aurantium var. sinensis, Eucalyptus grandis, Glycine max, Chlorella variabilis, Leucaena glauca, Lotus japonicus, Medicago tribuloides, Mimulus guttatus, Musa malaccensis, Oryza sativa, Panicum italicum, Physcomitrium patens, Phaseolus vulgaris, Pisum sativum, Populus balsamifera, Populus trichocarpa, Ricinus communis, Selaginella moellendorffii, Sisymbrium salsugineum, Solanum lycopersicum, Theobroma cacao, Triticum aestivum, Vitis vinifera, Zea mays.

Nicotiana benthamiana

10 or 20 µl of freshly prepared total protein from Arabidopsis thaliana suspension culture (A), 10 or 20 µl of total protein (older extract) from Arabidopsis thaliana suspension culture (B), 10 or 20 µl of total leaf extract from Nicotiana tabacum (C), 10 or 20 µl of freshly prepared total protein from Nicotiana tabacum leaf protoplasts (D), 10 or 20 µl total protein from older extract of Nicotiana tabacum leaf protoplasts (E) , extracted with buffer containing 100 mM Tris (pH 7.8), 200 mM NaCl, 1 mM EDTA, 2% (v/v) beta-mercaptoethanol and 0.2% (v/v) Triton X-100, were separated on 8% SDS-PAGE and blotted 2h to nitrocellulose (semidry blot at 200 mA and RT). Blots were blocked with 5% (w/v) milk powder and 1% (w/v) BSA over night at 4°C with agitation. Blot was incubated in the primary antibody at a dilution of 1:2 000 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 HRP conjugated) diluted to 1:10 000 in blocking solution for 45 min. 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 120 seconds.

Courtesy Fabian Künzl, University of Tuebingen, Germany

~10 µg (lane 1) and ~50 µg (lane 2) of Chlamydomonas reinhardtii whole-cell lysates were resolved by SDS-PAGE and transferred to PVDF membrane. Anti-clathrin heavy chain antibody (Agrisera AS10 690) was diluted 1:2500 in 5% (w/v) milk + 1% (w/v) fish skin gelatin in TBST and incubated overnight at 4° C. After washing and incubating with HRP-conjugated secondary antibody, diluted 1:10,000 for 30 minutes at room temperature, the membrane was washed and developed with chemiluminescent substrate of extreme low femtogram range,  for 5 minutes following the manufacturer’s instructions. Exposure time on film was 5 seconds.

Courtesy of Dr. Branch Craige, University of Massachusetts Medical School, USA

 


Cells were fixed with 4% paraformaldehyde, permeabilized, blocked in 3% fish skin gelatin +1% BSA in PBST, and incubated with anti-clathrin heavy chain antibody (diluted 1:2500, Agrisera AS10 690) and anti-acetylated tubulin (diluted 1:2000, Sigma T6793) antibody diluted in block overnight at 4° C. After washing and labeling with fluorescent secondary antibodies (Molecular Probes; clathrin: Alexa 488, green in the image; acetylated tubulin: Alexa 568, red in the image), the coverslips were mounted on Prolong Gold Anti-Fade (Molecular Probes) and imaged by widefield epifluorescence. Scale bar = 5 µm.

Courtesy of Dr. Branch Craige, University of Massachusetts Medical School, USA

Clathrin is a protein involved in intracellular trafficking and plays a major role in the formation of coated vesicles. It consists of three clathrin heavy chains and three light chains. Clathrin-coated vesicles (CCV) selectively sort cargo at the cell membrane, trans-Golgi network, and endosomal compartments for multiple membrane traffic pathways.