All lanes : Anti-CRISPR-Cas9 antibody [7A9-3A3] (ab191468) at 5 µg/ml

Lane 1 : S2 non-transfected cell lysate
Lane 2 : S2 cells transfected with CRISPR-Cas9 plasmid

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 160 kDa
Observed band size: 160 kDa


Exposure time: 4 minutes

We recommend using 3% milk as the blocking agent in Western Blot.

ab191468 stained in NIH3T3 cells. Untreated and Cas9 transfected cells were fixed with 4% paraformaldehyde (10min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab191468 at 10µg/ml and ab6046 (Rabbit polyclonal to beta tubulin) at 1ug/ml overnight at +4°C. The secondary antibodies were ab150117 (colored green) used at 1 ug/ml and ab150087 (pseudo-colored red) used at 2ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.

Lane 1: HeLa inducible Cas9 cell line in the absence of Doxcycline (-DOX)

Lane 2: HeLa inducible Cas9 cell line in the treated for 24 hours in the presence of 1ug/ml of Doxcycline (+DOX).

Blot was probed with Anti-CRISPR-Cas9 antibody [7A9-3A3] (ab191468) 1:2000 and Anti-Tubulin Antibody DM1A (Sigma) as loading control. Shows specific band at 170kDA in Dox treated cells and tubulin band at 50kDa. Lysis and running Cells lysed in 1.5X Lammeli buffer +0.15M DTT syringed 10X with 25g needle and then boiled 100 degrees C for 10 minutes. 10ug of sample run out on 4-12% Bis Tris Nupage gel in 1X MOPS buffer WB protocol Western blot was performed by wet transfer of gel onto nitrocellulose membrane 100V for 2 h 4 degrees. Blocked in 5% milk PBST for 1 h room temp (1X PBS + 0.1% tween 20) incubated 24h at 4 degrees in 5% PBST with primary antibodies 1:2000 Anti-CRISPR-Cas9 antibody [7A9-3A3] (ab191468) as well as 1:3000 Anti Tubulin Mouse monoclonal DM1A antibody ( Sigma) Washed 3X PBST IgG anti mouse HRP 1:5000 (Jackson immunoscience) Washed 3x PBST Washed

HeLa cell line contains Cas9 under the control of a Doxcycline/Tetracycline inducible promotor (Tet on) Specific band at 170 kDa appears upon treatment with Doxcycline 1ug/ml for 24hours.1X PBS Developed using ECL prime kit (GE healthcare).