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73211
Cas9 and Associated Proteins Antibody Sampler Kit

Cas9 and Associated Proteins Antibody Sampler Kit #73211

Western Blotting Image 1

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Cas9 (S. pyogenes) (+), using Cas9 (S. pyogenes) Rabbit mAb mAb (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 2

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Cas9 (S. aureus), Cas9 (S. pyogenes), AsCpf1 (Strain BV3L6), or FnCpf1 (Strain U112) (+), using Cas9 (S. aureus) (E4G3U) Rabbit mAb (upper) and Myc-Tag (7110) Rabbit mAb #2278 (lower).

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Western Blotting Image 3

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing AsCpf1 (Strain BV3L6), FnCpf1 (Strain U112), Cas9 (S. pyogenes), or Cas9 (S. aureus) (+), using AsCpf1 (Strain BV3L6) (E1U7C) Rabbit mAb (upper) and Myc-Tag (71D10) Rabbit mAb #2278 (lower).

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Western Blotting Image 4

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing FnCpf1 (Strain U112), AsCpf1 (Strain BV3L6), Cas9 (S. pyogenes), or Cas9 (S. aureus) (+), using FnCpf1 (Strain U112) (E7I2B) Rabbit mAb (upper) and Myc-Tag (71D10) Rabbit mAb #2278 (lower).

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Western Blotting Image 5

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

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Flow Cytometry Image 6

Flow cytometric analysis of 293T cells, untreated (blue) or transfected with S. pyogenes plasmid (green), using Cas9 (S. pyogenes) (D8Y4K) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

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Flow Cytometry Image 7

Flow cytometric analysis of 293T cells, untransfected (blue) or transfected with a construct expressing myc-tagged Cas9 (S. aureus) (green), using Cas9 (S. aureus) Rabbit mAb (solid line) compared to a concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

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Flow Cytometry Image 8

Flow cytometric analysis of 293 cells, untransfected (blue) or transfected with myc-tagged AsCpf1 (Strain BV3L6) (green), using AsCpf1 (Strain BV3L6) (E1U7C) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

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Flow Cytometry Image 9

Flow cytometric analysis of 293 cells, untransfected (blue) or transfected with a construct expressing myc-tagged FnCpf1 (Strain U112) (green), using FnCpf1 (Strain U112) (E7I2B) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

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IF-IC Image 10

Confocal immunofluorescent analysis of Cas9-expressing 293T cells (left) and unmodified 293T cells (right) using Cas9 (S. pyogenes) (D8Y4K) Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red).

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IF-IC Image 11

Confocal immunofluorescent analysis of 293T cells transfected with a construct expressing myc-tagged Cas9 (S. aureus) using Cas9 (S. aureus) (E4G3U) Rabbit mAb (green) and Myc-Tag (9B11) Mouse mAb #2276 (red). Overlapping signals are shown in yellow. Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IF-IC Image 12

Confocal immunofluorescent analysis of 293T cells transfected with a construct expressing myc-tagged AsCpf1 (Strain BV3L6) using AsCpf1 (Strain BV3L6) (E1U7C) Rabbit mAb (green) and Myc-Tag (9B11) Mouse mAb #2276 (red).

Overlapping signals are shown in yellow. Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IF-IC Image 13

Confocal immunofluorescent analysis of 293T cells transfected with a construct expressing myc-tagged FnCpf1 (Strain U112) using FnCpf1 (Strain U112) (E7I2B) Rabbit mAb (green) and Myc-Tag (9B11) Mouse mAb #2276 (red). Overlapping signals are shown in yellow. Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Cas9 (S. pyogenes) (D8Y4K) Rabbit mAb 65832 20 µl
  • WB
  • IF
  • F
All 150 Rabbit IgG
Cas9 (S. aureus) (E4G3U) Rabbit mAb 51610 20 µl
  • WB
  • IF
  • F
124 Rabbit IgG
AsCpf1 (Strain BV3L6) (E1U7C) Rabbit mAb 19984 20 µl
  • WB
  • IF
  • F
151 Rabbit IgG
FnCpf1 (Strain U112) (E7I2B) Rabbit mAb 90111 20 µl
  • WB
  • IF
  • F
152 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The Cas9 and Associated Proteins Antibody Sampler Kit provides an economical means of detecting Cas9 and Cas9-related family members. The kit includes enough antibody to perform two western blot experiments with each primary antibody.

Each antibody in the Cas9 and Associated Proteins Antibody Sampler Kit recognizes transfected levels of its target protein. Antibodies are specific for the indicated endonuclease target and cross-reactivity with other endonucleases is not observed.

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Val16 of Cas9 (S. pyogenes), Val905 of Cas9 (S. aureus), Leu822 of Acidaminococcus sp. Cpf1 (Strain BV3L6), and Ile841 of Cpf1 from Francisella tularensis subsp. novicida (Strain U112).

CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) are RNA-guided nuclease effectors that are utilized for precise genome editing in mammalian systems (1). Class 2 CRISPR systems rely on single-component effector proteins to mediate DNA interference (2). Several Class 2 CRISPR effector proteins, derived from specific bacterial species, are used for genome editing. Cas9 family of proteins, derived from S. pyogenes and S. aureus, are some of the most well characterized and widely used editing effector enzymes. Additional members of the Class2 CRISPR system include Cpf1 (CRISPR from Prevotella and Francisella) endonucleases (3). Cpf1 endonucleases, compared to Cas9 systems, have several unique features that increase the utility of CRISPR-based genome editing techniques: 1) Cpf1-mediated cleavage relies on a single and short CRISPR RNA (crRNA) without the requirement of a trans-activating crRNA (tracrRNA), 2) Cpf1 utilizes T-Rich protospacer adjacent motif (PAM) sequences rather than a G-Rich PAM, and 3) Cpf1 generates a staggered, rather than a blunt-ended, DNA double-stranded break (3). These features broaden the utility of using CRISPR-Cas systems for specific gene regulation and therapeutic applications. Several Cpf1 bacterial orthologs, e.g. Francisella novicida U112 and Acidaminococcus sp. BV3L6, have been characterized for CRISPR-mediated mammalian genome editing (3, 4).

  1. Cong, L. et al. (2013) Science 339, 819-23.
  2. Horvath, P. and Barrangou, R. (2010) Science 327, 167-70.
  3. Zetsche, B. et al. (2015) Cell 163, 759-71.
  4. Zhang, Y. et al. (2017) Sci Adv 3, e1602814.
Entrez-Gene Id
901176
Swiss-Prot Acc.
U2UMQ6 , J7RUA5 , Q99ZW2 , A0Q7Q2
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

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