Immunofluorescent analysis of DNMT1/DNA Methyltransferase 1 was performed using 70% confluent log phase PANC-1 cells treated with 100ng of Interleukin-6 for 30 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with DNMT1/DNA Methyltransferase 1 Rabbit Polyclonal Antibody (PA1-880) at 2 ug/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (A27034) a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing predominantly nuclear localization. Panel e is untreated cell with predominantly cytoplasmic signal. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
|Tested species reactivity||Human, Rat|
|Published species reactivity||Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic peptide corresponding to residues A(171) K G P A K R K P Q E E S E(185) of human Dnmt1.|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS with 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunocytochemistry (ICC)||2 µg/mL|
|Immunofluorescence (IF)||2 µg/mL|
|Western Blot (WB)||2 µg/mL|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
|Western Blot (WB)||See 1 publications below|
PA1-880 detects DNA methyltransferase 1 (Dmnt1) from HeLa cell nuclear extracts.
PA1-880 has been successfully used in Western blot procedures. By Western blot, this antibody detects an ~180 kDa protein representing Dnmt1 from HeLa cell nuclear extract. A doublet between 40 and 45 kDa is also seen on Western blots using this antibody.
The PA1-880 immunogen is a synthetic peptide corresponding to residues A(171) K G P A K R K P Q E E S E(185) of human Dnmt1. PA1-880 immunizing peptide (Cat. # PEP-136) is available for use in neutralization and control experiments.
DNA methyltransferases (Dnmts) methylate the 5-position of cytosine in the context of CpG dinucleotides. DNA methylation is crucial for normal embryonic development, genetic imprinting, and X-chromosome inactivation. Hypermethylation of DNA has been implicated in tumorigenesis through improper methylation of growth regulatory gene promoter regions. There exist three isoforms of Dnmts, Dnmt1, 2 and 3. Dnmt1 is believed to perform most of the maintenance and de novo methylation activities that occur in somatic cells of mammals. Studies have shown that Dnmt1 can establish repression of transcription complexes consisting of Dnmt1, histone deacetylase 2 (HDAC2), and DNA methyltransferase 1 associated protein (DMAP1). Therefore, in addition to DNA methylation maintenance, Dnmt1 is thought to directly target transcriptionally repressive chromatin to the genome during S-phase DNA replication.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Expression of an alternative Dnmt1 isoform during muscle differentiation.
PA1-880 was used in western blot to investigate a DNA methyltransferase 1 isoform's expression level during the differentiation of muscle
|Aguirre-Arteta AM,Grunewald I,Cardoso MC,Leonhardt H||Cell growth and differentiation : the molecular biology journal of the American Association for Cancer Research (11:551)||2000|