CRISPR-Cas9 Antibody - BSA Free

Western Blot: CRISPR-Cas9 Antibody [NBP3-05547] - Western blot analysis of HEK293 cell lysates using chicken pAb to CRISPR-Cas9 [1] protein standard (red), [2] non-transfected cells and [3] transfected cells with GFP-CRISPR-Cas9 (C-terminal 803-1053 amino acids of CRISPR-Cas9) fusion construct. The band at about 53kDa corresponds to the GFP-CAS9 fusion protein.

Immunocytochemistry/Immunofluorescence: CRISPR-Cas9 Antibody [NBP3-05547] - HEK293 cells were transfected with a construct including the N-terminal 608 amino acids of S. aureus CRISPR-Cas9 fused to GFP and stained with CRISPR-Cas9 Antibody in red. Transfected cells express the green fusion protein and bind the antibody in red, producing a yellow signal. Nuclear DNA in transfected and non-transfected cells is revealed with the blue DNA stain DAPI.

• Reactivity: Ba
• Applications: WB, ICC/IF, IHC
• Clonality: Polyclonal
• Host: Chicken
• Conjugate: Unconjugated
• Format: BSA Free

CRISPR-Cas9 Antibody - BSA Free Summary

• Description: This antibody is supplied as a concentrated total IgY preparation derived from egg yolk. Production involves an initial organic extraction to remove lipids and lipoproteins, followed by salt fractionation of the aqueous phase to precipitate and enrich the IgY fraction. The material is then extensively dialyzed against PBS, and preservative is added during final formulation.
  
  Please note that the exact concentration of target-specific IgY cannot be determined, as the final preparation contains both antigen-specific IgY and non-immune IgY.
• Immunogen: C-terminal region of S. aureus CRISPR-Cas9 expressed in and purified from E. coli.
• Isotype: IgY
• Clonality: Polyclonal
• Host: Chicken
• Purity: IgY purified

Applications/Dilutions

• Dilutions:
  • Immunocytochemistry/ Immunofluorescence 1:1000-1:5000
  • Immunohistochemistry 1:1000-1:5000
  • Western Blot 1:1000
• Theoretical MW: 124 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Packaging, Storage & Formulations

• Storage: Store at 4C short term. For extended storage, add an equal volume of glycerol, aliquot and store at -20C or below. Avoid repeated freeze-thaw cycles.
• Buffer: Supplied as a concentrated total IgY preparation from egg yolk, dialyzed against PBS with added preservative.
• Preservative: 0.02% Sodium Azide
• Purity: IgY purified

Notes

Chicken products cannot be exported to Canada.

Alternate Names for CRISPR-Cas9 Antibody - BSA Free

• Cas9
• CRISPR
• CRISPR/Cas9
• CRISPR-associated endonuclease Cas9/Csn1
• CRISPR-associated protein 9 nuclease
• CRISPR-Cas9
• CRISPR-Cas9/Csn1
• csn1
• SPy_1046
• SPy1046
• SpyCas9

Background

Clustered regularly interspaced short palindromic repeats (CRISPRs) are derived from DNA fragments of bacteriophages that infect prokaryotes. When infected, the bacteria capture snips of DNA from the invading virus to create CRISPR arrays. During subsequent infections, the bacteria produce RNA segments from the CRISPR arrays to target the virus' DNA. CRISPR-associated protein 9 (Cas9) is RNA-guided, binds DNA, and is a cleaving enzyme that functions as an integral component of the bacterial CRISPR adaptive immune system that targets the virus' DNA to disable it (1). To check for sites complementary to the 20 base pair spacer region of the guide RNA (gRNA) of the CRISPR, Cas9 unwinds foreign DNA that invades the bacteria. If the DNA substrate is complementary to the gRNA, Cas9 cleaves the invading DNA, rendering the virus disabled. The presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the target DNA (protospacer) is required for Cas9 cleavage of foreign DNA. As PAM is absent in bacterial CRISPR loci, cleavage of the host genome is avoided and provides a novel sequence for identification of foreign DNA by Cas9.

Using CRISPR-Cas9 technology, double-stranded DNA breaks may be induced within specific targeted genome sequences (target DNA; protospacer) for insertion or removal of DNA sequences for gene editing applications. To target a specific loci, a gRNA that will bind to a specific target sequence of DNA within a genome is created. The gRNA will recognize the DNA sequence, and the Cas9 enzyme will cleave the DNA at the targeted location. Once the targeted DNA is removed by Cas9, the cell's own DNA repair mechanism is used to insert or remove a DNA sequence for genomic editing.

Cas9 detection is used to confirm and evaluate CRISPR Cas9 gRNA transfection efficiency. Western blot analysis of CRISPR-Cas9 gRNA transfected cell lysates with Cas9 antibodies identifies the protein having a theoretical molecular weight of 160kDa. Broad areas of research are benefiting from CRISPR-Cas9 based gene editing tools including studies of basic immunity functions, genetic screening and disease treatment (2). Ethical concerns have led to many countries making it illegal to manipulate human germline cells or perform embryo genome editing.

References

1. Oakes, B. L., Fellmann, C., Rishi, H., Taylor, K. L., Ren, S. M., Nadler, D. C., . . . Savage, D. F. (2019). CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification. Cell, 176(1-2), 254-267.e216. doi:10.1016/j.cell.2018.11.052

2. Chiou, S. H., Winters, I. P., Wang, J., Naranjo, S., Dudgeon, C., Tamburini, F. B., . . . Winslow, M. M. (2015). Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing. Genes Dev, 29(14), 1576-1585. doi:10.1101/gad.264861.115

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.