Revision 9

#19526

Store at -20C

CST Logo
Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Applications:
W, W-S, IHC-P, IF-IC, FC-FP

Reactivity:
All

Sensitivity:
Transfected Only

MW (kDa):
150

Source/Isotype:
Rabbit IgG

UniProt ID:
#Q99ZW2

Entrez-Gene Id:
901176

Product Usage Information

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:50 - 1:250
Immunohistochemistry (Paraffin) 1:400
Immunofluorescence (Immunocytochemistry) 1:100
Flow Cytometry (Fixed/Permeabilized) 1:800

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

For a carrier free (BSA and azide free) version of this product see product #98605.

Specificity/Sensitivity

Cas9 (S. pyogenes) (E7M1H) Rabbit mAb recognizes transfected levels of total Cas9 (S. pyogenes) protein. This antibody does not cross-react with Cas9 (S. aureus), FnCpf1, and AsCpf1 proteins.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu833 of Cas9 (S. Pyogenes) protein.

Background

The CRISPR associated protein 9 (Cas9) is an RNA-guided DNA nuclease and part of the Streptococcus pyogenes CRISPR antiviral immunity system that provides adaptive immunity against extrachromosomal genetic material (1). The CRISPR antiviral mechanism of action involves three steps: (i), acquisition of foreign DNA by host bacterium; (ii), synthesis and maturation of CRISPR RNA (crRNA) followed by the formation of RNA-Cas nuclease protein complexes; and (iii), target interference through recognition of foreign DNA by the complex and its cleavage by Cas nuclease activity (2). The type II CRISPR/Cas antiviral immunity system provides a powerful tool for precise genome editing and has potential for specific gene regulation and therapeutic applications (3). The Cas9 protein and a guide RNA consisting of a fusion between a crRNA and a trans-activating crRNA (tracrRNA) must be introduced or expressed in a cell. A 20-nucleotide sequence at the 5' end of the guide RNA directs Cas9 to a specific DNA target site. As a result, Cas9 can be "programmed" to cut various DNA sites both in vitro and in cells and organisms. CRISPR/Cas9 genome editing tools have been used in many organisms, including mouse and human cells (4,5). Research studies demonstrate that CRISPR can be used to generate mutant alleles or reporter genes in rodents and primate embryonic stem cells (6-8).

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

W: Western Blotting W-S: Simple Western™ IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized)

Cross-Reactivity Key

All: All Species Expected

Trademarks and Patents

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

Alexa Fluor is a registered trademark of Life Technologies Corporation.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 9

Cell Signaling Technology Logo
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Cas9 (S. pyogenes), using Cas9 (S. pyogenes) (E7M1H) XP® Rabbit mAb (upper) and Myc-Tag (71D10) Rabbit mAb #2278 (lower).
Western Blotting Image 1: Cas9 (<i>S. pyogenes</i>) (E7M1H) XP<sup>®</sup> Rabbit mAb
Simple Western™ analysis of lysates (0.001 mg/mL) from COS-7 cells transfected with a construct expressing Cas9 (S. pyogenes) cells using Cas9 (S. pyogenes) (E7M1H) XP® Rabbit mAb #19526. The virtual lane view (left) shows a single target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 66-440 kDa separation module.
Western Blotting Image 1: Cas9 (<i>S. pyogenes</i>) (E7M1H) XP<sup>®</sup> Rabbit mAb
Immunohistochemical analysis of Cas9 (S. pyogenes) expression in paraffin-embedded Nprl2-deficient 293 cell pellet (left, positive) or untreated 293 cell pellet (right, negative) using Cas9 (S. pyogenes) (E7M1H) XP® Rabbit mAb. Nprl2 expression was knocked out in the Nprl2-deficient cells by transient transfection of Cas9 (S. pyogenes) and Nprl2-specific guide sequences. 293/Nrpl2 -/- cells were kindly provided by Rachel Wolfson and Lynne Chantranupong of MIT, Cambridge, MA.
Immunohistochemistry Image 1: Cas9 (<i>S. pyogenes</i>) (E7M1H) XP<sup>®</sup> Rabbit mAb
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 9

Cell Signaling Technology Logo
Immunohistochemical analysis of Cas9 expression in paraffin-embedded Cas9 (S. Pyogenes) transgenic mouse brain (top-left, positive) and wild type mouse tissues (negative): brain (top-center), colon (top-right), liver (bottom-left), lung (bottom-center), and pancreas (bottom-right), using Cas9 (S. pyogenes) (E7M1H) XP® Rabbit mAb.
Immunohistochemistry Image 2: Cas9 (<i>S. pyogenes</i>) (E7M1H) XP<sup>®</sup> Rabbit mAb
Confocal immunofluorescent analysis of 293T cells transiently transfected with a myc-tagged Cas9 (S. pyogenes) construct, using Cas9 (S. pyogenes) (E7M1H) XP® Rabbit mAb (green) and Myc-Tag (9B11) Mouse mAb #2276 (red). Colocalization of green and red signals appear yellow in the composite image (bottom-right). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Immunofluorescence Image 1: Cas9 (<i>S. pyogenes</i>) (E7M1H) XP<sup>®</sup> Rabbit mAb
Flow cytometric analysis of 293 cells, untransfected (blue) or transfected with Cas9 (S. pyogenes) (green), using Cas9 (S. pyogenes) (E7M1H) XP® Rabbit mAb (solid lines) or concentraion-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 secondary antibody.
Flow Cytometry Image 1: Cas9 (<i>S. pyogenes</i>) (E7M1H) XP<sup>®</sup> Rabbit mAb
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.