HSP70 | Heat shock protein 70 (cytoplasmic)

70 kDa

Application example

1µg of total protein from Horderum vulgare pre heat shock leaf (1), Horderum vulgare post heat shock (2h 40ºC) (2), Zea mays pre heat shock total protein leaf (3), Zea mays post heat shock (2h 40ºC) (4), total protein leaf extracted with Agrisera Protein Eextraction Buffer (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 with chemiluminescent detection reagent with extreme femtogram range.

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, The Hebrew University of Jerusalem, Israel
 

200 fmoles of HSP70 protein standard product number AS08 371S (1), 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 Agrisera 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 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/RT  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 chemiluminescence detection reagent in extreme femtogram range,  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.

Can be sold containing 0.1% ProClin if requested

This antibody can be used as a marker of cytoplasmic fraction in tomato (Anfoka et al. 2015).

Applied primary antibody dilution in western blot depends upon sensitivity of detection reagents (pico or femtogram for chemiluminescent detection).

Immunoprecipitation protocol using Agrisera anti-Hsp70 cytosolic antibodies, see tab: protocols.