Description
Development of a multicellular organism from a single fertilized cell is regulated by the coordinated activity of DNA transcription factors. Oct3/4, a member of the POU family of transcription factors, functions in pluripotent cells of early embryonic stem cell (ES) lines and embryonal carcinomas (EC). Other members of the POU family include Oct1, Oct2, Pit-1, and unc-86. The POU domain, a 150-amino acid region that determines binding specificity, is conserved among these proteins and consists of 3 subdomains: POU-specific A and B subdomains and a homeobox-like subdomain. Oct3/4 is expressed in undifferentiated cells, but is lost as cells are induced to differentiate. Oct3/4 is not expressed in adult tissues. The interaction of Oct3/4 with SOX2, another embryonic transcription factor, produces an active complex that regulates expression of genes such as Nanog, UTF1, and FGF4. Although Oct3/4 is specifically phosphorylated on serine residues, this modification is not required for DNA binding, but may affect its transactivation potential. Thus, Oct3/4 is a transcription factor that plays an important role in determining early steps of embryogenesis and differentiation.
Bioimaging 1. Seed the cells in appropriate culture medium at an appropriate cell density in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219), and culture overnight to 48 hours. 2. Remove the culture medium from the wells, wash the wells twice with 100 µl of 1x PBS, and fix the cells by adding 100 µl of fresh 3.7% Formaldehyde in PBS or BD Cytofix™ fixation buffer (Cat. No. 554655) to each well and incubating for 10 minutes at room temperature (RT). 3. Remove the fixative from the wells, and wash the wells twice with 100 µl of 1x PBS. 4. Permeabilize the cells by adding 100 µl of 1× BD Perm/Wash™ buffer (Cat. No. 554723) to each well and incubating for 30 minutes at RT. 5. Remove the permeabilizer, and wash the wells twice with 100 µl of 1x PBS. 6. Dilute the antibody in BD Perm/Wash™ buffer, and stain the cells by adding 50 µl of the diluted antibody to each well and incubating for 1 hour at RT. 7. Remove the diluted antibody, and wash the wells three times with 100 µl of 1x PBS. 8. Remove the PBS, dilute the second-step reagent in BD Perm/Wash™ buffer, and stain the cells by adding 50 µl of the diluted second-step reagent to each well and incubating for 1 hour at RT. 9. Remove the diluted second-step reagent, and wash the wells twice with 100 µl of 1x PBS. 10. Remove the PBS, and counter-stain the nuclei by adding 100 µl of a 2 µg/ml solution of Hoechst 33342 (eg, Sigma-Aldrich Cat. No. B2261) in 1x PBS to each well at least 15 minutes before imaging. 11. View and analyze the cells on an appropriate imaging instrument.
Store undiluted at -20°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
4. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
