ENCBS853PVZ / cell line

Summary

Status
released
Term name
WTC11
Summary
Homo sapiens WTC11 cell line genetically modified (insertion) using CRISPR targeting H. sapiens SMAD4

Attribution

ENCODE4 project
Lab
Richard Myers, HAIB
Award PI
Richard Myers, HAIB
Submitted by
Scott Newberry
Source
Richard Myers
Project
ENCODE
External resources
Aliases
richard-myers:WTC11-3x FLAG-SMAD4-1

Genetic modifications

Accession
Category
Purpose
Method
Nucleic acid delivery method
Site
ENCGM925RYIinsertiontaggingCRISPR

Donor information

Status
released
Accession
ENCDO882UJI
Aliases
encode:donor of WTC11
Species
Homo sapiens
Life stage
adult
Age
30 years
Sex
male
Ethnicity
Japanese
External resources

Documents

immunoblot
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 negative control IPs prepared with K562 nuclear lysate (Lane 3) and WTC-11 nuclear lysate (Lane 5).
Submitted by
Scott Newberry
Lab
Richard Myers, HAIB