ENCBS619TUZ / 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 JUN

Accession	Category	Purpose	Method	Nucleic acid delivery method	Site
ENCGM300SHU	insertion	tagging	CRISPR		Target JUN-human

Caption excerpt: Each FLAG-tagged sample was immunoprecipitated from its corresponding nuclear…

Caption: Each FLAG-tagged sample was immunoprecipitated from its corresponding nuclear protein isolate (500 uL) using the FLAG Immunoprecipitation Kit (Sigma-Aldrich; cat# FLAGIPT1). The final elution step was performed by suspending the sample-bound resin in NuPage Sample Reducing Agent 10X and NuPage LDS Sample Buffer 4X (Thermo Fisher Scientific) and heating for 3 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. 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). The second western blot image depicts a negative control IP prepared with K562 nuclear lysate (Lane 3).
Submitted by: Scott Newberry
Lab: Richard Myers, HAIB
Grant: UM1HG009411