Cas9 | CRISPR-associated endonuclease 9 (polyclonal)
AS16 3690 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Cas9 from Streptococcus thermophilus
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1 : 1000 (WB)
|Expected | apparent MW||
Depends upon a MW of a protein which is a fusion partner.
|Not reactive in|
To be added when available, antibody released in March 2017.
Nicotiana benthamiana (N b.): sample from 5 weeks old plants second leaves (previously infiltrated with A.tumefaciens containing Cas9 transformation casettes(Nb+). Hordeum vulgare (Hv 1-4 , Hv- samples): 2 months old plants’ youngest leaves (stably transformed (Hv 1, -2, -3, -4) and wild type (Hv-) plants). 100 mg plant tissue (homogenised in liquid N2) +400 μl extraction buffer (15 min 4 °C 1500rpm centrifugation). Tissue was extracted with following extaction buffer: 10 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, 10 V% glicerol, 0,5 V% Nonidet P-40, 5 mM NaF. Right before use: 1 mM DTT, 100 mM PMSF, 200 mM Na3VO4 is added to the extraction buffer. Denaturation: sample in extraction buffer + equal volume 2X Laemmli buffer, 72°C, 10 min.
20 μl of the samples were separated on 8% SDS-PAGE, and blotted with transblot turblot turbo (BioRad) to 0.45 um pore size PVDF (Hybond, GE). Blots were blocked with 5% milk powder in PBST for 60 min at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 for 160 min at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 2 times for 10 min in PBST buffer at RT with agitation. Blot was incubated in secondary antibody (Goat anti-rabbit from Agrisera, AS09 602) diluted to 1:20 000 in PBST for 90 min at RT with agitation. The blot was washed as above and developed for 1 min with Bio-Rad Clarity Western ECL substrate using Bio-Rad ChemiDocTM Imaging System. Exposure time was 170.6 sec.
Courtesy Dr. Éva Hamar, ABC, Hungary