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Clathrin heavy-chain 1,2

AS10 690  |  Clonality: Polyclonal  |  Host: Rabbit  |  Reactivity: Arabidopis thaliana, Chlamydomonas reinhardtii, Nicotiana tabacum

Clathrin heavy-chain 1,2  in the group Antibodies for Plant/Algal  / Membrane Transport System / Endomembrane system at Agrisera AB (Antibodies for research) (AS10 690)

DATA SHEET IN PDF

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Product Information

Immunogen

KLH-conjugated peptide derived from available plant clathrin heavy chain sequences including Arabidopsis thaliana clathrin heavy chain 1 UniProt: Q0WNJ6, TAIR:At3g11130, clathrin heavy chain 2 UniProt: Q0WLB5,TAIR:At3g08530

Host Rabbit
Clonality Polyclonal
Purity Affinity purified serum in PBS, pH 7.4
Format Lyophilized
Quantity 100 µg
Reconstitution For reconstitution add 100 µl of sterile water.
Storage Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.
Tested applications Immunolocalization (IL), Immunofluorescence (IF), Western blot (WB)
Recommended dilution 1 : 2400 (IL), 1 :400 (IF), 1 : 2000 (WB)
Expected | apparent MW

193 | 170 kDa (Arabidopsis thaliana)

Reactivity

Confirmed reactivity Arabidopsis thaliana, Chlamydomonas reinhardtii, Nicotiana tabacum
Predicted reactivity

Amborella trichopoda, Brassica napus, Capsella rubella, Citrus aurantium var. sinensis, Eucalyptus grandisGlycine max, Chlorella variabilisLeucaena glauca, Lotus japonicusMedicago tribuloidesMimulus guttatus, Musa malaccensisOryza sativa, Panicum italicum, Physcomitrella patens, Phaseolus vulgaris, Pisum sativum, Populus balsamifera, Populus trichocarpa, Ricinus communis, Selaginella moellendorffii, Sisymbrium salsugineum, Solanum lycopersicum, Theobroma cacao, Triticum aestivum, Vitis vinifera


Species of your interest not listed? Contact us
Not reactive in

Nicotiana benthamiana

Application examples

Application examples Application example

western blot using anti-plant clathrin heavy chain antibody

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

 

western blot using anti-clathrin heavy chain antibody on Chlamydomonas samples    ~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

 

 immunolocalization using anti-clathrin heavy chain antibody   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

Additional information

Please, note that fresh samples will provide better restuls (see image below).

Related products

Related products

AS10 680 | Anti-Tubulin alpha chain, rabbit antibodies

AS10 681 | Anti-Tubulin beta chain, rabbit antibodies

Plant and algal protein extraction buffer

Secondary antibodies

Background

Background

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.

 

Product citations

Selected references Ranjan et al. (2019). Transient Internalization and Microtubule-Dependent Trafficking of a Ciliary Signaling Receptor from the Plasma Membrane to the Cilium. Curr Biol. 2019 Aug 2. pii: S0960-9822(19)30867-X. doi: 10.1016/j.cub.2019.07.022.
Wattelet-Boyer et al. (2016). Enrichment of hydroxylated C24- and C26-acyl- chain sphingolipids mediates PIN2 apical sorting at trans-Golgi network subdomains. Nat Commun. 2016 Sep 29;7:12788. doi: 10.1038/ncomms12788.
Derbyshire et al. (2015). Proteomic Analysis of Microtubule Interacting Proteins over the Course of Xylem Tracheary Element Formation in Arabidopsis. Plant Cell. 2015 Oct 2. pii: tpc.15.00314.
Grones et al. (2015). Auxin-binding pocket of ABP1 is crucial for its gain-of-function cellular and developmental roles. J Exp Bot. 2015 Apr 28. pii: erv177.
McLoughlin et al. (2013). Identification of novel candidate phosphatidic acid binding proteins involved in the salt stress response of Arabidopsis thaliana roots. Biochem J. Jan 17.

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