Goat anti-Rabbit IgG (H&L), HRP conjugated-free sample
AS09 602-free | clonality: polyclonal | host: goat | reactivity: rabbit IgG (H&L)
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1: 50 000 -1: 90 000 (ELISA), 1 : 75 000 with enhanced ECL and 1: 25 000 with regular ECL (WB), 1: 500 -1: 5000 (IHC)
|Selected references||MacLellan et al. (2015). Chaperone roles for TMAO and HSP70 during hyposmotic stress in the spiny dogfish shark (Squalus acanthias). J Comp Physiol B. 2015 Jun 7.
Giuntoli et al. (2014). A trihelix DNA binding protein counterbalances hypoxia-responsive transcriptional activation in Arabidopsis. PLoS Biol. 2014 Sep 16;12(9):e1001950. doi: 10.1371/journal.pbio.1001950. eCollection 2014.
Zubo et al. (2014). Inhibition of the electron transport strongly affects transcription and transcript levels in Arabidopsis mitochondria. Mitochondrion. 2014 Mar 31. pii: S1567-7249(14)00037-3. doi: 10.1016/j.mito.2014.03.011
Ivanov et al. (2014). SORTING NEXIN1 Is Required for Modulating the Trafficking and Stability of the Arabidopsis IRON-REGULATED TRANSPORTER1. Plant Cell. 2014 Mar 4.
5 µg of total extract from (1) Hordeum vulgaretotal leaf, (2) Zea mays (3) Spinacia oleracea extracted with PEB (AS08 300) were separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% ECL Advance blocking reagent (GE Healthcare) 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 anti-PsaC antibody (AS04 042) at a dilution of 1: 10 000 for 1h at room temperature 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 room temperature with agitation. Blots were incubated in secondary antibody (goat anti-rabbit IgG horse radish peroxidase conjugated, AGRISERA) diluted to 1:50 000 in 2% ECL Advance blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL Advance detection reagent according to the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad). Exposure time was 30 seconds.
Comparison of Agrisera secondary antibody sensitivity
10 μg of mitochondrial fraction from Arabidopsis thaliana (1,3) and Arabidopsis thaliana leaf extract (2,4) were separated on 10% gel and blotted on nitrocellulose membrane using wet transfer (0.22% CAPS, pH 11). Filters where blocked (1.5h) in 5% milk in TBST (1X TBS, 0,1% Tween 20), incubated with 1: 1000 anti-COXII antibodies (2h in TBST) followed by incubation with 1: 10 000 secondary anti-rabbit (1h) HRP-coupled antibodies from Agrisera (left panel) and other manufacture (right panel) and visualized with standard ECL on Kodak autoradiography film for 5 s. Antibody in left panel detects target protein also in total cell extract (2) and can be used in higher dilution than applied 1: 10 000.
Agrisera goat anti-rabbit HRP conjugated antibody (AS09 602) can be used at following dilutions: 1: 50 000 -1: 90 000 (ELISA), 1 : 75 000 with enhanced ECL and 1: 25 000 with regular ECL (WB), 1: 500 -1: 5000 (IHC).
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