ENCBS004EDA / cell line
Summary
- Status
- released
- Term name
- HepG2
- Term ID
- EFO:0001187
- Summary
- Homo sapiens HepG2 cell line genetically modified (insertion) using CRISPR targeting H. sapiens PHF21A
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:HepG2-3xFLAG-PHF21A-1
Genetic modifications
Accession | Category | Purpose | Method | Nucleic acid delivery method | Site |
|---|---|---|---|---|---|
| ENCGM448SCH | insertion | tagging | CRISPR |
|
Donor information
- Status
- released
- Accession
- ENCDO000AAC
- Aliases
- encode:donor of HepG2, bradley-bernstein:Donor of HepG2
- Species
- Homo sapiens
- Life stage
- child
- Age
- 15 years
- Sex
- male
- Health status
- hepatocellular carcinoma
- Ethnicity
- European
- External resources
- References
Documents
immunoblot
Caption excerpt: PHF21A Western (Lane 9). Predicted size was 78 kDa. The observed size was 120…
- Caption
- PHF21A Western (Lane 9). Predicted size was 78 kDa. The observed size was 120 kDa, which is within 20% of an observed band of 100 kDa seen in https://www.novusbio.com/products/phf21a-antibody_nbp2-04043. 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 HepG2 nuclear lysate (Lane 6).
- Submitted by
- Scott Newberry
- Lab
- Richard Myers, HAIB
- Grant
- UM1HG009411