ENCBS392EUI / cell line
Summary
- Status
- released
- Term name
- K562
- Term ID
- EFO:0002067
- Summary
- Homo sapiens K562 cell line genetically modified (insertion) using CRISPR targeting H. sapiens FOXA3
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:K562-3x FLAG-FOXA3-2
Genetic modifications
Accession | Category | Purpose | Method | Nucleic acid delivery method | Site |
---|---|---|---|---|---|
ENCGM965KHZ | insertion | tagging | CRISPR |
|
Donor information
- Status
- released
- Accession
- ENCDO000AAD
- Aliases
- encode:donor of K562, bradley-bernstein:Donor of K562 cells
- Species
- Homo sapiens
- Life stage
- adult
- Age
- 53 years
- Sex
- female
- Health status
- chronic myelogenous leukemia (CML)
- External resources
- References
Documents
immunoblot
- Caption
- Each FLAG-tagged sample was immunoprecipitated from its corresponding nuclear protein isolate (500 uL - nuclear and 1 mL - cytoplasmic) 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 M2Peroxidase-conjugated ANTI-FLAG antibody (diluted in 5% BSA solution) (SigmaAldrich; 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 HepG2 nuclear lysate (Lane 2), HepG2 cytoplasmic lysate (Lane 6), K562 nuclear lysate (Lane 3), and WTC-11 nuclear lysate (Lane 5).
- Submitted by
- Scott Newberry
- Lab
- Richard Myers, HAIB
- Grant
- UM1HG009411