ENCBS456DNR / cell line

Status: released
Term name: K562
Term ID: EFO:0002067
Summary: Homo sapiens K562 cell line genetically modified (insertion) using CRISPR targeting H. sapiens GATA2

Accession	Category	Purpose	Method	Nucleic acid delivery method	Site
ENCGM676SHN	insertion	tagging	CRISPR		Target GATA2-human

Caption excerpt: GATA2 Western (Lane 8). Expected size: 54 kDa. Each nuclear protein isolate…

Caption: GATA2 Western (Lane 8). Expected size: 54 kDa. Each nuclear protein isolate (50 mcg) was standardized in a solution containing a volume of 2% Halt Protease and Phosphatase Inhibitor Single-Use Cocktail Mixture (Thermo Fisher Scientific), NuPage Sample Reducing Agent 10X, and NuPage LDS Sample Buffer 4X (Thermo Fisher Scientific). After heating the solution for 15 minutes at 90C followed by cooling on ice, the protein samples were loaded onto a NuPage 4-12% Bis-Tris gel (Thermo Fisher Scientific) and separated using a PowerEase 90W system (Thermo Fisher Scientific) running at 150 V for 1 hour. A HepG2 untagged nuclear isolate was included as a negative control, and a ZNF7-tagged nuclear isolate as a positive control. The protein bands were transferred to a nitrocellulose membrane using the Invitrogen iBlot 2 System (Thermo Fisher Scientific), and blocked overnight at 4C in 5% milk solution with gentle rocking. The membrane was treated with a 1:5000 dilution of monoclonal M2-Peroxidase-conjugated ANTI-FLAG antibody (diluted in 5% BSA solution) (Sigma-Aldrich; cat# A8592) for 1 hour. Following four 5-minute washes with 1X TBST, visualization was attained with the Super Signal West Femto solution kit (Thermo Fisher Scientific) and a MyECL Imager (Thermo Fisher Scientific).
Submitted by: Scott Newberry
Lab: Richard Myers, HAIB
Grant: UM1HG009411