Immunofluorescent analysis of BMAL1 was performed using 70% confluent log phase SH-SY5Y cells. 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 BMAL1 Rabbit Polyclonal Antibody (PA1-523) 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 nuclear localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Hamster, Human, Mouse, Rat|
|Published species reactivity||Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic Peptide: C D(582) M I D N D Q G S S S P S(594)|
|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)||1:500-1:2000|
* 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.
PA1-523 detects BMAL1/aryl hydrocarbon nuclear translocator 3 (ARNT3) from hamster and mouse tissues as well as recombinant human BMAL1.
PA1-523 has been sucessfully used in Western blot procedures. By Western blot, this antibody detects a 110 kDa protein which corresponds to the product of a hamster GST-BMAL1 fusion construct overexpressed in E. coli. This antibody detects a non-specific band in U87-MG cell lysates at ~105kDa and in NIH-3T3 cell lysates at ~135kDa.
PA1-523 immunizing peptide corresponds to amino acid residues 582-594 from mouse BMAL1. This sequence is completely conserved between mouse, rat, guinea pig, and human BMAL1. PA1-523 immunizing peptide (Cat. # PEP-075) is available for use in neutralization and control experiments.
Circadian rhythmicity is a basic property of phylogenetically diverse organisms which range from animals and plants, to fungi. Regulation of endogenous biological clocks is regulated at the genetic level by a protein-mediated, autoregulatory feed-back loop. In mammals, several genes that encode members of the basic helix-loop helix (bHLH) PAS (PER-ARNT-SIM) transcription factor family have been shown to play a significant role in regulating circadian oscillations. Transactivation of CLOCK-induced genes is mediated via an E box enhancer (CACGTG) found upstream of target genes. CLOCK-ARNT3 heterodimers bind to E box regulatory elements and stimulate gene transcription. CLOCK has been shown to transactivate the mammalian homolog of Drosophila per. PER, in concert with the product of the mammalian timeless gene (TIM), negatively regulates its own transcription by blocking the activity of the CLOCK-BMAL1 transactivation complex.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
The transcription factor Runx2 is under circadian control in the suprachiasmatic nucleus and functions in the control of rhythmic behavior.
PA1-523 was used in western blot to study the circadian control of Runx2 expression in the suprachiasmatic nucleus and its role in regulating circadian behavioural rhythms
|Reale ME,Webb IC,Wang X,Baltazar RM,Coolen LM,Lehman MN||PloS one (8:null)||2013|
|Mouse||1:200 to 1:1,000||
Expression of circadian rhythm genes in gonadotropin-releasing hormone-secreting GT1-7 neurons.
PA1-523 was used in western blot to investigate the role of the clock machinery in GnRH neuronal function.
|Gillespie JM,Chan BP,Roy D,Cai F,Belsham DD||Endocrinology (144:5285)||2003|
Circadian Transcription. Thinking outside the E-Box.
PA1-523 was used in immunocytochemistry to study the role of E-Box in the pathway of circadian clock.
|Muñoz E,Brewer M,Baler R||The Journal of biological chemistry (277:36009)||2002|