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Cas9 (S. pyogenes) Antibody [L14C20]

Cat.No.: F0428

Application: Reactivity: All Species Expected

Usage Information

Dilution
WB1:1000 IF1:100 - 1:400 FCM1:50 - 1:200

Application
WB, IF, FCM

Reactivity
All Species Expected

Source
Mouse Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
160 kDa

Datasheet & SDS

Biological Description

Specificity
Cas9 (S. pyogenes) Antibody [L14C20] detects endogenous levels of total Cas9 (S. pyogenes) protein.

Clone
L14C20

Synonym(s)
cas9; CRISPR-associated endonuclease Cas9/Csn1; SpCas9; SpyCas9; csn1; SPy_1046

Background
Cas9 from Streptococcus pyogenes (SpCas9) is a 1,368-amino-acid RNA-guided endonuclease central to type II CRISPR adaptive immunity, forming a ribonucleoprotein complex with a guide RNA (gRNA: crRNA-tracrRNA hybrid) that recognizes and cleaves invading viral DNA at NGG protospacer adjacent motif (PAM) sites, a property that has made SpCas9 foundational for precise genome editing applications. SpCas9 adopts a bilobal architecture: an N-terminal recognition (REC) lobe composed of REC1 and REC2 domains that cradles the gRNA for target scanning, and a C-terminal nuclease (NUC) lobe housing the RuvC (residues 1–59, 718–769, 1096–1184) and HNH (residues 775–908) endonuclease domains, separated by a helical PAM-interrogating bridge helix (Arg1333/Glu1219). Additional L1 and L2 structural lobes stabilize the central channel through which DNA threads after seed-pairing. The gRNA-Cas9 complex initially scans DNA for PAM sequences via shallow minor groove contacts (Arg1335/Gln1119), then initiates base-pairing from the PAM-proximal “seed” region (positions 1–12), displacing the non-target DNA strand to form an R-loop hybrid stabilized by a hydrogen-bonding network (notably Ser808/Arg661 with the phosphate backbone). This triggers a conformational switch that opens the HNH and RuvC catalytic centers, each coordinated by two Mg²⁺ ions: HNH (Arg661/Asp839) cleaves the target strand, and RuvC (Asp10/His983/Asp986) nicks the non-target strand, resulting in a blunt double-strand break (DSB) 3 bp upstream of the PAM via sequential cleavage (RuvC before HNH, with a ~0.2s delay). Fidelity is ensured by seed mismatch destabilization and PAM-distal proofreading. SpCas9’s programmable nature enables not only genome editing, but also base editing (e.g., dCas9-cytosine deaminase fusions) and transcriptional regulation (e.g., dCas9-VP64 for activation/repression), with therapeutic applications such as correction of sickle cell disease through BCL11A editing.

Background

Cas9 from Streptococcus pyogenes (SpCas9) is a 1,368-amino-acid RNA-guided endonuclease central to type II CRISPR adaptive immunity, forming a ribonucleoprotein complex with a guide RNA (gRNA: crRNA-tracrRNA hybrid) that recognizes and cleaves invading viral DNA at NGG protospacer adjacent motif (PAM) sites, a property that has made SpCas9 foundational for precise genome editing applications. SpCas9 adopts a bilobal architecture: an N-terminal recognition (REC) lobe composed of REC1 and REC2 domains that cradles the gRNA for target scanning, and a C-terminal nuclease (NUC) lobe housing the RuvC (residues 1–59, 718–769, 1096–1184) and HNH (residues 775–908) endonuclease domains, separated by a helical PAM-interrogating bridge helix (Arg1333/Glu1219). Additional L1 and L2 structural lobes stabilize the central channel through which DNA threads after seed-pairing. The gRNA-Cas9 complex initially scans DNA for PAM sequences via shallow minor groove contacts (Arg1335/Gln1119), then initiates base-pairing from the PAM-proximal “seed” region (positions 1–12), displacing the non-target DNA strand to form an R-loop hybrid stabilized by a hydrogen-bonding network (notably Ser808/Arg661 with the phosphate backbone). This triggers a conformational switch that opens the HNH and RuvC catalytic centers, each coordinated by two Mg²⁺ ions: HNH (Arg661/Asp839) cleaves the target strand, and RuvC (Asp10/His983/Asp986) nicks the non-target strand, resulting in a blunt double-strand break (DSB) 3 bp upstream of the PAM via sequential cleavage (RuvC before HNH, with a ~0.2s delay). Fidelity is ensured by seed mismatch destabilization and PAM-distal proofreading. SpCas9’s programmable nature enables not only genome editing, but also base editing (e.g., dCas9-cytosine deaminase fusions) and transcriptional regulation (e.g., dCas9-VP64 for activation/repression), with therapeutic applications such as correction of sickle cell disease through BCL11A editing.

References
https://pubmed.ncbi.nlm.nih.gov/24505130/ https://pubmed.ncbi.nlm.nih.gov/24476820/

Reference Table for Selecting PVDF Membrane Pore Size Specifications

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Handling Instructions

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%