AS13 2640 Anti-ACT | Actin (polyclonal)

Confirmed reactivity:	Agostis stolonifera cv. ‘Penncross’,Arabidopsis thaliana, Brassica napus, Cucumis sativus, Cyanthobasis fruticulosa, Cynara cardunculus, Fragaria x ananassa,, Glycine max, Hordeum vulgare, Nicotiana tabacum, Odontarrhena lesbiaca, Petrosimonia nigdeensis, Phaseolus vulgaris, Phaeodactylum tricornutum, Phoenix dactylifera, Picrorhiza kurroa,Salsola grandis, Salsola tragus, Setaria italica, Solanum tuberosum, Triticum aestivum, Vigna unguiculata, Zea mays
Predicted reactivity:	Agropyron cristatum, Beta vulgaris, Betula luminifera, Brassica rapa subsp. pekinensis, Daucus carota, Cannabis sativa L. , Capsella rubella,Castanea sativa, Chorispora bungeana, Cyanidioschyzon merolae strain 10D,  Glycine soja, Halogeton glomeratus, Helianthus annuus, Ipomoea batatas, Manihot esculenta, Medicago truncatula, Malus domestica, Oryza sativa, Pisum sativun, Populus sp., Saccharum officinarum, Solanum lycopersicum, Solanum tuberosum, Phaeodactylum tricornutum, Picea abies, Picea sitchensis, Prunus avium, Olea europaea, Ricinus communis, Rubus plicatus, Theobroma cacao, Trebouxia sp., Vicia faba Species of your interest not listed? Contact us
Not reactive in:	Chlamydomonas reinhardtii (too high background for this species)

15 µg of total protein extracted with PEB (AS08 300) from  leaf tissue of (1) Arabidopsis thaliana, (2) Hordeum vulgare, (3) Zea mays were separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Filters were blocked 1h with 2% low-fat milk powder in TBS-T (0.1% TWEEN 20) and probed with anti-actin (AS13 2640, 1:2500, 1h) and secondary anti-rabbit (1:10 000, 1 h) antibody (HRP conjugated, recommended secondary antibody AS09 602) in TBS-T containing 2% low fat milk powder. Antibody incubations were followed by washings in TBS-T (15, +5, +5, +5 min). All steps were performed at RT with agitation. Signal was detected with chemiluminescent detection reagent using a Fuji LAS-3000 CCD (300s, standard sensitivity). Exposure time was 2 min.

 

Type of material: isolated plant protoplasts from Arabidopsis thaliana wild type
Fixation: formaldehyde | Methanol, 37°C | 3.2 % paraformaldehyde in W5 buffer (150 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES pH 5.7), fixed overnight at +4ºC Hydrophilization: no
Cell wall digestion: yes 
Membrane permeabilization: DMSO-IGEPA
Antigen retrieval: no
Blocking buffer: 2% BSA in 1X Phosphate Buffered Saline (PBS) buffer pH 7.2
Washing buffer: after primary antibody incubation 3 washes were done with 2% BSA in 1X PBS buffer, after secondary antibody incubation – with 1X PBS buffer
Primary antibody dilution and incubation time: 1:250 in blocking buffer, incubation was done overnight at +4ºC
Secondary antibody dilution and incubation time and supplier: anti-rabbit antibody conjugated with DyLight™ 594 (AS12 2076, Agrisera), diluted in a blocking buffer to a final concentration of 1:500.
Incubation time: 3 hours at room temperature on a shaker.
Co-staining of the nucleus (DAPI): no
Cell wall and nucleus staining: no 

Details of the ExM (Expansion microscopy method) on A. thaliana and Zea mays protoplasts are described here.

Courtesy Dr. Kirk Czymmek, The Donald Danforth Plant Science Center, USA

 

Actin cytoskeleton in 5 days old Arabidopsis thaliana seedlings. Actin signal shown in green, PIN1 in red and DAPI in blue. The material has been fixed in 2 % formaldehyde for 45 minutes. Tissue cleaning has been performed before immunolocalization. Rabbit anti-actin primary antibody was diluted in 1:250 and anti-rabbit Alexa 488 and Alexa 555 were both diluted in 1: 500 (Invitrogen). Scale bar - 20 µm.

Courtesy: Dr. Taras Pasternak, Freiburg University, Germany

 

Proteins were extracted from tuber flesh of Russet Burbank potato (Solanum tuberosum) with 0.1 M Tris HCl (pH=8.0), 5% sucrose (m/v), 2% (m/v) SDS, protease inhibitors (PMSF 1mM). Samples were heated 95°C 5 min, and 10 μg of total protein was resolved in 12% SDS PAGE and blotted to PVDF membrane for 1h-1.5h using tank transfer. Blots were blocked with a skimmed milk 4% (m/v) in T-TBS (1.5h) at RT with agitation. Primary antibodies (AS13 2640) were applied overnight +4°C in dilution 1:5000 with agitation. After washing with T-TBS 2-3 times, membrane was incubated with secondary antibodies (Goat Anti-Rabbit HRP conjugate, Transgen biotech HS101) 1:10000 for 1 hour at RT. Blot was washed as above and developed with ECL (Clarity Western ECL Substrate, BioRad, 170-5060) for 5 – 10 minutes. Exposure time – 20.395 seconds.

Courtesy of Iauhenia Isayenka, University of Sherbrooke, Canada

Antibody available in 3 various pack sizes: 50, 100 and 150 µl - Please inquire.

This product can be sold containing ProClin if requested.

Actin is a highly conserved protein and an essential component of cell cytoskeleton and plays an important role in cytoplasmic streaming, cell shape determination, cell division, organelle movement and extension growth. Preferentially expressed in young and expanding tissues, floral organ primordia, developing seeds and emerging inflorescence.

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Agrisera Western Blot protocol and video tutorials

Protocols to work with plant and algal protein extracts