D366-3
Lot 003~
Page 1For Research Use Only.
Not for use in diagnostic procedures.

Anti-SaCas9 mAb
CODE No.

D366-3

CLONALITY
CLONEISOTYPE
QUANTITY

Monoclonal
11E7-9
Mouse IgG2a 
100 L, 1 mg/mL

SOURCEIMMUNOGEN
REACTIVITY

Purified IgG from hybridoma supernatant
Recombinant protein, corresponding to amino acids 1-462 of Staphylococcus aureus Cas9
This clone specifically reacts with Staphylococcus aureus Cas9 (SaCas9) and does notcross-reacts with Streptococcus pyogens Cas9 (SpCas9).
PBS containing 50% Glycerol (pH 7.2). No preservative is contained.
This antibody solution is stable for one year from the date of purchase when stored at -20°C.

FORMULATIONSTORAGEAPPLICATIONS-CONFIRMED
Western blotting
Immunoprecipitation
Immunocytochemistry

0.5 g/mL
1 g/sample
1 g/mL

SPECIES CROSS REACTIVITY on WB
Species

Human

Mouse

Rat

Staphylococcus aureus

Cell

Not tested

Not tested

Not tested

Transfectant

Reactivity



REFERENCE

1) Ran, F. A., et al., Nature 520, 186-191 (2015)

For more information, please visit our website at https://ruo.mbl.co.jp/.

URL https://ruo.mbl.co.jpe-mail support@mbl.co.jp

D366-3
Lot 003~
Page 2The descriptions of the following protocols are examples. Each user should determine the appropriate condition.

SDS-PAGE & Western blotting1) Wash the cells 3 times with PBS and suspends them in 1 mL of Laemmli’s sample buffer, then sonicate briefly (up to 10 sec.).
2) Boil the samples for 3 min. and centrifuge. Load 10 L of the sample per lane in a 1-mm-thick SDS-polyacrylamide gel(7.5% acrylamide) for electrophoresis.
3) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hr. in a semi-dry transfer system(Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% methanol). See the manufacturer's manual for precise transferprocedure.
4) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS) overnight at 4°C.
5) Incubate the membrane with primary antibody diluted with 1% skimmed milk (in PBS) as suggested in the APPLICATIONS
for 1 hr. at room temperature. (The concentration of antibody will depend on the conditions.)
6) Wash the membrane with PBS-T [0.05% Tween-20 in PBS] (3 times for 5 min.).
7) Incubate the membrane with 1:10,000 of Anti-IgG (Mouse) pAb-HRP (MBL, code no. 330) diluted with 1% skimmed milk(in PBS) for 1 hr. at room temperature.
8) Wash the membrane with PBS-T (3 times for 5 min.).
9) Wipe excess buffer on the membrane, and then incubate it with appropriate chemiluminescence reagent for 1 min. Remove
extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap.
10) Expose to an X-ray film for 1 min. in a dark room. Develop the film as usual. The condition for exposure and development
may vary.
(Positive control for Western blotting; Transfectant)Western blotting analysis of SaCas9
Lane 1: 293T
Lane 2: SaCas9-DN-HA/293T
Lane 3: SaCas9-WT-HA/293T
Immunoblotted with Anti-SaCas9 mAb (MBL, code no. D366-3)
Samples were kindly provided by Dr. Haruhiko Siomi.
(Department of Molecular Biology, Keio University School of Medicine)D366-3
Lot 003~Page 3

Immunoprecipitation
1) Wash 1 x 107 cells 3 times with PBS and suspend them with 2 mL of Extraction buffer [50 mM Tris-HCl (pH 7.4), 150 mM
NaCl, 0.05% NP-40] containing appropriate protease inhibitors, then sonicate briefly (up to 15 sec.).
2) Incubate the tube for 15 min. on ice.
3) Centrifuge the tube at 12,000 x g for 5 min. at 4°C and transfer the supernatant to another tube.
4) Mix 20 L of 50% protein A agarose beads slurry resuspended in 200 L of IP buffer [10 mM Tris-HCl (pH 8.0), 500 mM
NaCl, 0.1% NP-40] with primary antibody as suggested in the APPLICATIONS. Incubate with gentle agitation for 1 hr. atroom temperature.
5) Centrifuge the tube at 2,500 x g for 10 seconds and discard the supernatant.
6) Resuspend the beads with 1 mL of IP buffer.
7) Centrifuge the tube at 2,500 x g for 10 seconds and discard the supernatant.
8) Repeat steps 6)-7) twice.
9) Add 200 L of cell lysate (prepared sample from step 3) into the tube. Incubate with gentle agitation for 1 hr. at room
temperature.
10) Centrifuge the tube at 2,500 x g for 10 seconds and discard the supernatant.
11) Resuspend the beads with 1 mL of Extraction buffer.
12) Centrifuge the tube at 2,500 x g for 10 seconds and discard the supernatant.
13) Repeat steps 11)-12) 5 times.
14) Resuspend the beads in 20 L of Laemmli’s sample buffer, boil for 3 min. and centrifuge.
15) Load 10 L per lane in a 1-mm-thick SDS-polyacrylamide gel (7.5% acrylamide) for electrophoresis.
16) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hr. in a semi-dry transfer system(Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% methanol). See the manufacturer's manual for precise transferprocedure.
17) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS) overnight at 4°C.
18) Incubate the membrane with primary antibody diluted with 1% skimmed milk (in PBS) as suggested in the APPLICATIONS
for 1 hr. at room temperature. (The concentration of antibody will depend on the conditions.)
19) Wash the membrane with PBS-T [0.05% Tween-20 in PBS] (3 times for 5 min.).
20) Incubate the membrane with 1:10,000 of Anti-IgG (Mouse) pAb-HRP (MBL, code no. 330) diluted with 1% skimmed milk(in PBS) for 1 hr. at room temperature.
21) Wash the membrane with PBS-T (3 times for 5 min.).
22) Wipe excess buffer on the membrane, and then incubate it with appropriate chemiluminescence reagent for 1 min. Remove
extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap.
23) Expose to an X-ray film for 1 min. in a dark room. Develop the film as usual. The condition for exposure and development
may vary.
(Positive control for Immunoprecipitation; Transfectant)Immunoprecipitation of SaCas9 from 293T transfectant
Sample: SaCas9-WT-HA/293T
Lane 1: Mouse IgG2a (isotype control) (MBL, code no. M076-3)
Lane 2: Anti-SaCas9 mAb (MBL, code no. D366-3)
Lane 3: Anti-HA-tag mAb (MBL, code no. M180-3)

Immunoblotted with Anti-SaCas9 mAb (MBL, code no. D366-3)
Sample was kindly provided by Dr. Haruhiko Siomi.
(Department of Molecular Biology, Keio University School of Medicine)D366-3
Lot 003~Page 4

Immunocytochemistry1) Spread the cells on a glass slide, then incubate in a CO2 incubator overnight.
2) Remove the culture supernatant by careful aspiration.
3) Wash the slide twice with PBS.
4) Fix the cells with 4% paraformaldehyde/PBS for 10 min. at room temperature (20~25°C).
5) Wash the slide twice with PBS.
6) Permeabilize the cells with 0.1% Triton X-100/PBS for 10 min. at room temperature.
7) Wash the slide twice with PBS.
8) Tip off PBS and add the primary antibody diluted with PBS as suggested in the APPLICATIONS onto the cells. Incubatefor 1 hr. at room temperature. (Optimization of antibody concentration or incubation condition is recommended if necessary.)9) Wash the slide 3 times with PBS.
10) Add 200 L of 1:500 Alexa Fluor® 488 Goat Anti-mouse IgG (Thermo Fisher Scientific, code no. A-11001) diluted with PBS
onto the cells. Incubate for 30 min. at room temperature. Keep out light by aluminum foil.
11) Wash the slide 3 times with PBS.
12) Wipe excess liquid from the slide but take care not to touch the cells. Never leave the cells to dry.
13) Counterstain with DAPI for 5 min. at room temperature.
14) Wash the slide once with PBS.
15) Promptly add mounting medium onto the slide, then put a cover slip on it.
(Positive control for Immunocytochemistry; Transfectant)Immunocytochemical detection of SaCas9 in HeLa transfectants
Right: SaCas9-WT-HA/HeLa
Left: SaCas9-DN-HA/HeLa
Green: Anti-SaCas9 mAb (MBL, code no. D366-3)
Blue: DAPI
Samples were kindly provided by Dr. Haruhiko Siomi.
(Department of Molecular Biology, Keio University School of Medicine)

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