Anti-CRISPR-Cas9 antibody [7A9-3A3]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CRISPR-Cas9 antibody [7A9-3A3] (AB191468)

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.

• WB

AbReview66778****

Western blot - Anti-CRISPR-Cas9 antibody [7A9-3A3] (AB191468)

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).

All lanes:

Western blot - Anti-CRISPR-Cas9 antibody [7A9-3A3] (ab191468)

Predicted band size: 160 kDa

false

This image was courtesy of an anonymous AbReview

• WB

Lab

Western blot - Anti-CRISPR-Cas9 antibody [7A9-3A3] (AB191468)

Gel type : MOPS

Blocking buffer : 3% milk block

All lanes:

Western blot - Anti-CRISPR-Cas9 antibody [7A9-3A3] (ab191468) at 5 µg/mL

Lane 1:

NIH 3T3 whole cell lysate at 20 µg

Lane 2:

NIH 3T3 overexpressing Cas9 whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 160 kDa

Observed band size: 160 kDa

false

Exposure time: 4min

• WB

Lab

Western blot - Anti-CRISPR-Cas9 antibody [7A9-3A3] (AB191468)

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

All lanes:

Western blot - Anti-CRISPR-Cas9 antibody [7A9-3A3] (ab191468) at 5 µg/mL

Lane 1:

S2 non-transfected cell lysate at 20 µg

Lane 2:

S2 cells transfected with CRISPR-Cas9 plasmid at 20 µg

Secondary

All lanes:

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

Predicted band size: 160 kDa

Observed band size: 160 kDa

true

Exposure time: 4min