HSP70 | Heat shock protein 70 (cytoplasmic)

AS08 371 | Clonality:Polyclonal | Host:Rabbit | Reactivity:A. thaliana, C. sativus, C. reinhardtii, D. subspicatus, E. tef, G. vermiculophylla, H. vulgare, M. sativa, P. strobus, S. vulgaris, S. lycopersicum, Trebouxia TR1 and TR9, Z. mays, P. falciparum

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Background		Heat-shock protein 70 (Hsp70) is the major stress-inducible protein in vertebrates and is highly conserved throughout evolution. It plays a role as a molecular chaperone and is important for allowing cells to cope with acute stressor insult, especially those affecting the protein machinery. Heat shock cognate protein 70 (HSC70), is a highly conserved protein and a member of the family of molecular chaperones.
Immunogen		KLH-conjugated synthetic peptide conserved in known higher plant HSC70 proteins including three isoforms of Arabidopsis thaliana HSC70-1 (NP_ 001119156.1), HSC70-2 (NP_195869.1) and HSC70-3 (NP_187555.1) as well as heat shock inducible Hsp70 of Arabidopsis thaliana AT3g12580/T2E22_110 and At1g16030 and AT3g12580/T2E22_110
Host		Rabbit
Clonality		Polyclonal
Clone
Purity		Serum
Format		Lyophilized
Quantity		50 µl
Reconstitution		For reconstitution add 50 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		Immunoprecipitation (IP), Western blot (WB)
Related products		AS08 348 \| anti-chloroplastic HSP70 AS08 347 \| anti-mitochondrial HSP70 Plant and algal protein extraction buffer Secondary antibodies
Additional information		Immunoprecipitation protocol using Agrisera anti-Hsp70 cytosolic antibodies can be found here.

application information
Recommended dilution		1 : 3000 5 µg protein/well (WB), 2-3 µl/protein extract of concentration 3-5 mg/ml
Expected \| apparent MW		70 kDa
Confirmed reactivity		Arabidopsis thaliana, Chlamydomonas reinhardtii, Chlamydomonas sp. UWO241, Cucumis sativus, E. teft, Hordeum vulgare, Medicago sativa, Silene vulgaris, Solanum lycopersicum, Pinus strobus, Polyscias elegans, Zea mays, algae: Desmodesmus subspicatus, Gracilaria vermiculophylla, phycobiont: Trebouxia TR1 and TR9, Plasmodium falciparum
Predicted reactivity		Ageratina adenophora, Allium sativum, Arabis alpina, Arachis diogoi, Arundo donax, Brassica napus, brassica rapa subsp. pekinensis, Camellia sinensis, Citrus sinensis, Coffea arabica, Eriobotrya japonica, Gossypium arboretum, Glycine max, Glycine soja, Hordeum vulgare var. distichum, Lotus japonicus, Medicago sativa, medicago truncatula, Musa acuminata subsp. malaccensis, Nicotiana tabacum, Nicotiana bethamiana, Oryza sativa, Phaseolus vulgaris, Physcomitrella patens, Pinus taeda, Pisum sativum, Populus balsamifera, Populus trichocarpa, Salix gilgiana, Saussurea medusa,Solanum tuberosum, Solanum commersonii, Spinacia oleracea, Tragopogon dubius, Tragopogon porrifolius, Triticum aestivum, Vitis vinifera, Volvox sp.
Not reactive in		No confirmed exceptions from predicted reactivity are currently known.
Additional information		This product can be sold containing ProClin if requested
Selected references		Barghetti et al. (2017). Heat-shock protein 40 is the key farnesylation target in meristem size control, abscisic acid signaling, and drought resistance. Genes Dev. 2017 Nov 15;31(22):2282-2295. doi: 10.1101/gad.301242.117. Gorovits et al. (2017). The six Tomato yellow leaf curl virus genes expressed individually in tomato induce different levels of plant stress response attenuation. Cell Stress Chaperones. 2017 Mar 21. doi: 10.1007/s12192-017-0766-0. Fernández-Bautista N. et al. (2017). AtHOP3, a member of the HOP family in Arabidopsis, interacts with BiP and plays a major role in the ER stress response. Plant Cell Environ. 2017 Feb 2. doi: 10.1111/pce.12927. Hammann et al. (2016). Selection of heat‑shock resistance traits during the invasion of the seaweed Gracilaria vermiculophylla. Marine Biology 163: 104. Shen et al. (2016). The Arabidopsis polyamine transporter LHR1/PUT3 modulates heat responsive gene expression by enhancing mRNA stability. Plant J. 2016 Aug 19. doi: 10.1111/tpj.13310. [Epub ahead of print] Gorovits et al. (2016). Tomato yellow leaf curl virus confronts host degradation by sheltering in small/midsized protein aggregates. Virus Res. 2016 Feb 2;213:304-13. doi: 10.1016/j.virusres.2015.11.020. Epub 2015 Dec 1. Ghandi et al. (2016). Tomato yellow leaf curl virus infection mitigates the heat stress response of plants grown at high temperature. Sci Rep. 2016 Jan 21;6:19715. doi: 10.1038/srep19715.

Application example

1µg of total protein from (1) Horderum vulgare pre heat shock leaf extracted with PEB (AS08 300), (2) Horderum vulgare post heat shock (2h 40ºC) leaf extracted with PEB (AS08 300), (3) Zea mays pre heat shock total protein leaf extracted with PEB (AS08 300), (4) Zea mays post heat shock (2h 40ºC) total protein leaf extracted with PEB (AS08 300) were separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF (Milipore). Filters were blocked 1h with 2% low-fat milk powder in TBS-T (0.1% TWEEN 20) and probed with anti-HSP70 antibody (AS08 371, 1:20 000, 1h) and secondary anti-rabbit (1:20 000, 1 h) antibody (HRP conjugated) in TBS-T containing 2% low fat milk powder. All steps were performed at RT with agitation. Signal was detected withECL Advance (GE Healthcare)

western blot detection using anti-hsp70 antibody

Protein from Solanum lycopersicum (1) total cell extract ca. 30 -50 µg, (2) and (3) nuclei pellet , (4) and (5) ca. 7 µg of nuclei fraction, (6) and (7) cytoplasmic pellet, (8) ca. 7 µg of cytoplasm fraction, were separated on 10% SDS-PAGE and blotted 1h to nitrocellulose (Schleicher & Schuell). Filters were blocked 1h with 2% low-fat milk powder in TBS-T (0.1% TWEEN 20) and probed with anti-HSP70 antibody (AS08 371, 1:5000, 3h RT). The antibody solution was decanted and the blot was rinsed briefly. Washed 3 times for 15 min in TBS-T at room temperature with agitation. Blot was incubated with a secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 5:000. The blot was washed as above and developed for 1 min with ECL detection reagent according to the manufacturers instructions.

Courtesy Dr Rena Gorovits

200 fmoles of HSP70 protein standard (1), product number AS08 371S, 1 µg of total protein from samples such as Lycopersicum esculentum leaf (2), Nicotiana tabaccum leaf, (3), Zea mays leaf (4), Hordeum vulgare leaf (5), Arabidopsis thaliana leaf (6) were extracted with Protein Extraction Buffer PEB (AS08 300). Samples were diluted with 1X sample buffer (NuPAGE LDS sample buffer (Invitrogen) supplemented with 50 mM DTT and heat at 70°C for 5 min and keept on ice before loading. Protein samples were separated on 4- 12% Bolt Plus gels, LDS-PAGE and blotted for 70 minutes to PVDF using tank transfer. Blots were blocked immediately following transfer in 2% blocking reagent (GE RPN 2125; Healthcare) or 5% non-fat milk dissolved in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 10 000 (in blocking reagent) for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, and then washed 1x15 min and 3x5 min with TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, recommended secondary antibody AS10 1489, Agrisera) diluted to 1:25 000 in blocking reagent for 1h at room temperature with agitation. The blots were washed as above. The blot was developed for 5 min with TMA-6 (Lumigen) detection reagent according the manufacturers instructions. Images of the blots were obtained using a CCD imager (VersaDoc MP 4000) and Quantity One software (Bio-Rad). Exposure time was 30 seconds.

western blot using plant anti-HSP70 antibodies

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Accessories: HSP70 | Heat shock protein 70 (cytoplasmic)

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