Components

• Guide-it Cas9 Monoclonal Antibody (Clone TG8C1)
  • Cat. # 632628: 100 µg Guide-it Cas9 Monoclonal Antibody (Clone TG8C1) (1 µg/µl)
  • Cat. # 632627:  3 x 100 µg Guide-it Cas9 Monoclonal Antibody (Clone TG8C1) (1 µg/µl)

• Guide-it Cas9 Polyclonal Antibody
  • Cat. # 632607: 100 µl Cas9 Polyclonal Antibody
  • Cat. # 632606:  3 x 100 µl Cas9 Polyclonal Antibody

Western blot analysis of the Guide-it Cas9 Monoclonal Antibody (Clone TG8C1)

The Guide-it Cas9 Monoclonal Antibody (Clone TG8C1), which was raised in mouse against recombinant full-length Cas9 (rCas9), was tested via Western blot analysis using either increasing concentrations of recombinant Cas9 (Panel A) or lysates made from cells transiently expressing Cas9 after plasmid transfection (Panel B). Panel A: Increasing concentrations of rCas9 (1 ng, 2 ng, 3 ng, and 5 ng) were analyzed via Western blotting. The samples were separated using a 7.5% SDS PAGE gel, followed by transfer onto a membrane. The membrane was probed with a 1:3,000 dilution of the monoclonal anti-Cas9 antibody and detection was performed using an anti-mouse HRP-coupled secondary antibody at a 1:10,000 dilution. A clearly visible band at the expected molecular weight of ~160 kDa was detected in each lane containing rCas9, even for the smallest amount of rCas9 loaded (1 ng). No signal was detected in the HEK 293 lane, which contains a lysate of 5,000 untransfected HEK 293 cells. Panel B: HEK 293 cells were transiently transfected with a plasmid encoding Cas9 protein. Lysates made from an increasing number of Cas9-expressing cells (1,000, 2,000, and 5,000 cells) were separated using a 7.5% SDS PAGE gel, followed by transfer onto a membrane. The membrane was probed with a 1:3,000 dilution of the monoclonal anti-Cas9 antibody and detection was performed using an anti-mouse HRP-coupled secondary antibody at a 1:10,000 dilution. A clearly visible band at ~160 kDa was detected only in the lanes containing lysates of transfected cells. No signal was detected in the HEK 293 lane, which contains a lysate of untransfected HEK 293 cells, and the NIH 3T3 lane, which contains a lysate of untransfected NIH 3T3 cells. The Marker lanes contain a molecular weight marker.

The Cas9 monoclonal antibody detects Cas9 protein by immunocytochemistry (ICC)

The Cas9 monoclonal antibody detects Cas9 protein by immunocytochemistry (ICC). RPE cells stably expressing ZsGreen1 were transfected with 0.35 µg of a plasmid encoding wild-type Cas9 under the control of the SV40 promoter using Xfect Transfection Reagent. Cells were fixed with 4% paraformaldehyde 48 hours post-transfection and stained with Cas9 monoclonal antibody (1:100) for one hour at room temperature (RT), followed by staining with donkey anti-mouse secondary antibody (1:1,000) for 30 minutes at RT. The antifade mounting media contained DAPI, allowing for nuclear staining.

The sensitivity of Cas9 polyclonal antibody assessed by Western blot

The sensitivity of Cas9 polyclonal antibody assessed by Western blot. Various amounts of recombinant Cas9 (rCas9) were loaded (Lane 3: 0.15 ng, Lane 4: 0.30 ng, Lane 5: 0.625 ng, Lane 6: 1.25 ng, Lane 7: 2.5 ng, Lane 8: 5.0 ng, Lane 9: 10.0 ng, Lane 10: 20.0 ng). Cell lysates prepared from 3.5 x 104 cultured cells (Lane 1: HT1080 cells, Lane 2: HEK 293 cells) were used as a negative control. The blot was probed with Cas9 polyclonal antibody (1:1,000 dilution). For detection, an HRP-conjugated goat anti-rabbit secondary antibody was used (1:5,000 dilution), and signal was detected using ECL reagent.

The specificity of Cas9 polyclonal antibody assessed by Western blot

The specificity of Cas9 polyclonal antibody assessed by Western blot. Lysates prepared from various untransfected cell lines (3.5 x 104 cultured cells; Lane 1: HepG2, Lane 2: CHO-K1, Lane 3: HeLa, Lane 4: MCF7, Lane 5: HEK 293, Lane 6: HT1080, Lane 7: NIH 3T3, Lane 8: no cells) and HEK 293 cells expressing Cas9 from the CMV promoter (stable cell line; Lane 10: 2.0 x 103 cells, Lane 11: 5.0 x 103 cells, Lane 12: 1.0 x 104 cells, Lane 13: 1.5 x 104 cells). One nanogram of recombinant Cas9 (rCas) was used as a positive control (Lane 9). The blot was probed with Cas9 polyclonal antibody (1:1,000 dilution). For detection, an HRP-conjugated goat anti-rabbit secondary antibody was used (1:5,000 dilution), and signal was detected using ECL reagent.

The Cas9 polyclonal antibody detects Cas9 protein by immunocytochemistry (ICC)

The Cas9 polyclonal antibody detects Cas9 protein by immunocytochemistry (ICC). RPE cells stably expressing ZsGreen1 were transfected with 0.35 µg of a plasmid encoding wild-type Cas9 under the control of the SV40 promoter using Xfect Transfection Reagent. Cells were fixed with 4% paraformaldehyde 48 hours post-transfection and stained with Cas9 polyclonal antibody (1:150) for one hour at room temperature (RT), followed by staining with donkey anti-rabbit secondary antibody(1:100) for 30 min at RT. In the control, cells were only stained with the secondary antibody.

The Cas9 polyclonal antibody recognizes wild-type and nickase mutant Cas9 protein

The Cas9 polyclonal antibody recognizes wild-type and nickase mutant Cas9 protein. HEK 293 cells were transfected with plasmids encoding either wild-type or mutant Cas9 transcripts expressed from the CMV promoter using Xfect Single Shots; untransfected cells were used as a negative control. After 48 hr, the cells were lysed and Western blotting was performed with 3.5x104 cells for the negative control and 0.7 x 104 cells for Cas9-expressing cells. Blots were probed with primary anti-Cas9 polyclonal antibody (1:5,000 dilution). For detection, an HRP-conjugated goat anti-rabbit secondary antibody was used (1:5,000 dilution), and signal was detected using ECL reagent. The image shown is a 0.5 sec exposure.