ENCBS546RGE / 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 BRCA1
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:K562-3xFLAG-BRCA1-1
Genetic modifications
Accession | Category | Purpose | Method | Nucleic acid delivery method | Site |
|---|---|---|---|---|---|
| ENCGM209JNV | 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 excerpt: BRCA1 Western (Lane 2). Predicted size was 211 kDa. The observed sizes were…
- Caption
- BRCA1 Western (Lane 2). Predicted size was 211 kDa. The observed sizes were 280 kDa, 230 kDa, and 115 kDa, which are within 20% of observed bands of 290 kDa, 220 kDa, and 130 kDa seen in https://www.abcam.com/brca1-antibody-ab238983.html. The dark band around 80 kDa is a non-distinct band seen in the K562 negative control. Each nuclear protein isolate (95 mcg - BRCA1, 78 mcg - TP53, 129 mcg - THAP7, 162 mcg - ZNF441, and 157 mcg - ZNF879) 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. 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 five 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 controls prepared with HepG2 nuclear lysate (Lane 2) and K562 nuclear lysate (Lane 3).
- Submitted by
- Scott Newberry
- Lab
- Richard Myers, HAIB
- Grant
- UM1HG009411