ENCBS589TUN / 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 NFAT5
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-NFAT5-1
Genetic modifications
Accession | Category | Purpose | Method | Nucleic acid delivery method | Site |
|---|---|---|---|---|---|
| ENCGM243SUD | 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: NFAT5 Western (Lane 7). Predicted size was 169 kDa. The observed size was 70…
- Caption
- NFAT5 Western (Lane 7). Predicted size was 169 kDa. The observed size was 70 kDa, which is within 20% of an observed band of 65 kDa seen in https://www.avivasysbio.com/nfat5-antibody-middle-region-arp37465-p050.html. The dark band around 125 kDa may correspond with one of the additional larger isoforms sharing the same tagged stop codon, and the faint banding may be due to degradation products. Each FLAG-tagged sample was immunoprecipitated from its corresponding protein isolate (1 mL) 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 cytoplasmic lysate (Lane 10).
- Submitted by
- Scott Newberry
- Lab
- Richard Myers, HAIB
- Grant
- UM1HG009411