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NLS-Cas9-EGFP

NLS‐Cas9‐EGFP-NLS is a fusion protein containing a nuclear localization sequence (NLS) on its N terminal and EGFP-NLS on the C terminal. 

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$242.00

Data sheet

Storage -70°C. Avoid freeze thaw cycles.
Background NLS‐Cas9‐EGFP-NLS is a fusion protein containing a nuclear localization sequence (NLS) on its N terminal and EGFP-NLS on the C terminal. The Cas9 RNP complex can localize to the nucleus immediately upon entering the cell. The EGFP can be taken as a reporter for tracking or sorting transfected cells, which creates the possibility of enriching cell populations for desired genome edits via fluorescence activated cell sorting (FACS).
Formulation Recombinant NLS-Cas9-EGFP-NLS protein expressed in E. coli supplied in a buffer of 10 mM Tris‐HCl (pH 7.4), 0.1 mM EDTA, 1 mM DTT, 300 mM NaCl, and 50% (v/v) Glycerol.
Concentration 50 μg/ 50 μl or 1 μg/μl or molar concentration 5.31 µM.

More info

For molar concentration and other related product information, please refer to:    CRISPR/Cas9 related enzyme proteins, antibodies, and other reagents


In vitro Cas9 mediated digestion of DNA

 

1. Add the following components to a sterile, nuclease-free tube on ice:

 

Components

Volume

Final Concentration

 

sgRNA (300 nM)

2 µl

~30 nM

 

Cas9-EGFP Nuclease Protein

(1000 nM)

0.60 µl

~30 nM

 

10X Cas9 Nuclease Reaction

Buffer

2 μl

1X

 

Nuclease-free H2O

12.4 μl

-

 

Pre-Incubate for 15 minutes at room temperature

 

Substrate DNA (30 nM)

3 μl

3 nM

 

Total Volume

20 μl

-

 

 

2. Collect all components by a brief centrifugation. Incubate the reaction at 37 °C for 1 hour.

3. Analyze fragments by running an agarose gel electrophoresis, or do transfection for target gene tracking.

 

 References:

1.  Deactivated CRISPR Associated Protein 9 for Minor-Allele Enrichment in Cell-Free DNA    Amin Aalipour et al. Clinical Chemistry 2018 Vol. 64, Issue 2 p307-p916
2. Purified Cas9 Fusion Proteins for Advanced Genome Manipulation Jovan Mircetic et al. Small Methods 2017 1, 1600052
3. Disruptive non-disruptive applications of CRISPR/Cas9  Jonathan LSchmid-Burgk  Current Opinion in Biotechnology December 2017, Volume 48 Pages 203-209
4. Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP  technology using recombinant CRISPR ribonucleoproteins Toshitsugu Fujita et al. Genes to Cells  Volume 21, Issue 4 April 2016  Pages 370–377
5. High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding Evan A Boyle  et al. PNAS 2017 May, 114 (21) 5461-5466
6.CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells  Wulan Deng et al. PNAS2015 vol. 112 | no. 38 p11870-11875

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