Cas9 | CRISPR-associated endonuclease 9 (monoclonal)
AS17 4124 | Clonality: Monoclonal | Host: Mouse | Reactivity: Cas9 from Streptococcus pyogenes
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Protein extracts from HEK293 cells were transfected with a myc-tagged Cas9. Visualization was made using monoclonal anti-Cas) antibodies used in annotated dilutions.
Protein samples were separated on 7.5 % SDS-PAGE and blotted 1h to nitrocellulose membrane. Blots were blocked with 3 % non-fat dry milk in PBS+Tween for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a given dilution for 1h at RT with agitation in PBS-T. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 in for 1h at RT with agitation. The blot was washed as above and incubated in ECL solution followed by exposure to an X-ray film.
Protein extracts from HEK293 cells were transfected with a myc-tagged Cas9. Transfected cells (lane 2 and 4) and untransfected cells (lane 1 and 3) cells. Detection using anti-myc antibody (lane 3 and 4).
IP was performed on whole cell extracts (100 μg) from HEK293 cells transfected with a Flag-tagged Cas9 using monoclonal anti-Cas9 antibodies. The immunoprecipitated proteins were subsequently analysed by Western blot with the antibody. Lane 3 and 4 show the result of the IP; a negative IP control (IP on untransfected cells) and the input (15 μg) are shown in lane 2 and 1, respectively.
Hela cells were transiently transfected with a Flag-tagged Cas9 expression vector. 48 hours post transfection the cells were fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton-X-100 and blocked in PBS containing 2% BSA for 2 hours at RT. The cells were stained with monoclonal anti-Cas9 (A and B) or with an anti- Flag (C) antibody at 4°C O/N, followed by incubation with an anti-mouse secondary antibody coupled to AF488 for 1 h at RT. Nuclei were counter-stained with Hoechst 33342 (bottom).
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