Overview

  • Product name
    Anti-BMAL1 antibody - ChIP Grade
    See all BMAL1 primary antibodies
  • Description
    Rabbit polyclonal to BMAL1 - ChIP Grade
  • Host species
    Rabbit
  • Specificity
    Detects BMAL 1 / aryl hydrocarbon nuclear translocator 3 (ARNT 3) from hamster tissues as well as recombinant human BMAL 1.
  • Tested applications
    Suitable for: ICC/IF, WB, IP, ChIPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Hamster, Human
    Predicted to work with: Horse, Chicken
  • Immunogen

    Synthetic peptide corresponding to Mouse BMAL1 aa 582-594.
    Sequence:

    DMIDNDQGSSSPS


    (Peptide available as ab4959)

Properties

Applications

Our Abpromise guarantee covers the use of ab3350 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF Use a concentration of 1 µg/ml.
EMSA Use at an assay dependent concentration. PubMed: 22210883
WB 1/200. Predicted molecular weight: 69.4 kDa.Can be blocked with BMAL1 peptide (ab4959). This antibody detects a protein which corresponds to the product of a hamster BMAL 1 fusion construct overexpressed in E. coli (110 kDa band)as well as recombinant human BMAL1.
IP Use at an assay dependent concentration. PubMed: 23547261
ChIP Use at an assay dependent concentration. PubMed: 18039858

Target

  • Function
    Transcriptional activator which forms a core component of the circadian clock. The circadian clock, an internal time-keeping system, regulates various physiological processes through the generation of approximately 24 hour circadian rhythms in gene expression, which are translated into rhythms in metabolism and behavior. It is derived from the Latin roots 'circa' (about) and 'diem' (day) and acts as an important regulator of a wide array of physiological functions including metabolism, sleep, body temperature, blood pressure, endocrine, immune, cardiovascular, and renal function. Consists of two major components: the central clock, residing in the suprachiasmatic nucleus (SCN) of the brain, and the peripheral clocks that are present in nearly every tissue and organ system. Both the central and peripheral clocks can be reset by environmental cues, also known as Zeitgebers (German for 'timegivers'). The predominant Zeitgeber for the central clock is light, which is sensed by retina and signals directly to the SCN. The central clock entrains the peripheral clocks through neuronal and hormonal signals, body temperature and feeding-related cues, aligning all clocks with the external light/dark cycle. Circadian rhythms allow an organism to achieve temporal homeostasis with its environment at the molecular level by regulating gene expression to create a peak of protein expression once every 24 hours to control when a particular physiological process is most active with respect to the solar day. Transcription and translation of core clock components (CLOCK, NPAS2, ARNTL/BMAL1, ARNTL2/BMAL2, PER1, PER2, PER3, CRY1 and CRY2) plays a critical role in rhythm generation, whereas delays imposed by post-translational modifications (PTMs) are important for determining the period (tau) of the rhythms (tau refers to the period of a rhythm and is the length, in time, of one complete cycle). A diurnal rhythm is synchronized with the day/night cycle, while the ultradian and infradian rhythms have a period shorter and longer than 24 hours, respectively. Disruptions in the circadian rhythms contribute to the pathology of cardiovascular diseases, cancer, metabolic syndromes and aging. A transcription/translation feedback loop (TTFL) forms the core of the molecular circadian clock mechanism. Transcription factors, CLOCK or NPAS2 and ARNTL/BMAL1 or ARNTL2/BMAL2, form the positive limb of the feedback loop, act in the form of a heterodimer and activate the transcription of core clock genes and clock-controlled genes (involved in key metabolic processes), harboring E-box elements (5'-CACGTG-3') within their promoters. The core clock genes: PER1/2/3 and CRY1/2 which are transcriptional repressors form the negative limb of the feedback loop and interact with the CLOCK
    NPAS2-ARNTL/BMAL1
    ARNTL2/BMAL2 heterodimer inhibiting its activity and thereby negatively regulating their own expression. This heterodimer also activates nuclear receptors NR1D1/2 and RORA/B/G, which form a second feedback loop and which activate and repress ARNTL/BMAL1 transcription, respectively. ARNTL/BMAL1 positively regulates myogenesis and negatively regulates adipogenesis via the transcriptional control of the genes of the canonical Wnt signaling pathway. Plays a role in normal pancreatic beta-cell function; regulates glucose-stimulated insulin secretion via the regulation of antioxidant genes NFE2L2/NRF2 and its targets SESN2, PRDX3, CCLC and CCLM. Negatively regulates the mTORC1 signaling pathway; regulates the expression of MTOR and DEPTOR. Controls diurnal oscillations of Ly6C inflammatory monocytes; rhythmic recruitment of the PRC2 complex imparts diurnal variation to chemokine expression that is necessary to sustain Ly6C monocyte rhythms. Regulates the expression of HSD3B2, STAR, PTGS2, CYP11A1, CYP19A1 and LHCGR in the ovary and also the genes involved in hair growth. Plays an important role in adult hippocampal neurogenesis by regulating the timely entry of neural stem/progenitor cells (NSPCs) into the cell cycle and the number of cell divisions that take place prior to cell-cycle exit. Regulates the circadian expression of CIART and KLF11. The CLOCK-ARNTL/BMAL1 heterodimer regulates the circadian expression of SERPINE1/PAI1, VWF, B3, CCRN4L/NOC, NAMPT, DBP, MYOD1, PPARGC1A, PPARGC1B, SIRT1, GYS2, F7, NGFR, GNRHR, BHLHE40/DEC1, ATF4, MTA1, KLF10 and also genes implicated in glucose and lipid metabolism. Represses glucocorticoid receptor NR3C1/GR-induced transcriptional activity by reducing the association of NR3C1/GR to glucocorticoid response elements (GREs) via the acetylation of multiple lysine residues located in its hinge region. Promotes rhythmic chromatin opening, regulating the DNA accessibility of other transcription factors. The NPAS2-ARNTL/BMAL1 heterodimer positively regulates the expression of MAOA, F7 and LDHA and modulates the circadian rhythm of daytime contrast sensitivity by regulating the rhythmic expression of adenylate cyclase type 1 (ADCY1) in the retina.
  • Tissue specificity
    Hair follicles (at protein level). Highly expressed in the adult brain, skeletal muscle and heart.
  • Sequence similarities
    Contains 1 bHLH (basic helix-loop-helix) domain.
    Contains 1 PAC (PAS-associated C-terminal) domain.
    Contains 2 PAS (PER-ARNT-SIM) domains.
  • Post-translational
    modifications
    Ubiquitinated, leading to its proteasomal degradation.
    O-glycosylated; contains O-GlcNAc. O-glycosylation by OGT prevents protein degradation by inhibiting ubiquitination. It also stabilizes the CLOCK-ARNTL/BMAL1 heterodimer thereby increasing CLOCK-ARNTL/BMAL1-mediated transcription of genes in the negative loop of the circadian clock such as PER1/2/3 and CRY1/2.
    Acetylated on Lys-538 upon dimerization with CLOCK. Acetylation facilitates CRY1-mediated repression. Deacetylated by SIRT1, which may result in decreased protein stability.
    Phosphorylated upon dimerization with CLOCK. Phosphorylation enhances the transcriptional activity, alters the subcellular localization and decreases the stability of the CLOCK-ARNTL/BMAL1 heterodimer by promoting its degradation. Phosphorylation shows circadian variations in the liver with a peak between CT10 to CT14. Phosphorylation at Ser-90 by CK2 is essential for its nuclear localization, its interaction with CLOCK and controls CLOCK nuclear entry.
    Sumoylated on Lys-259 upon dimerization with CLOCK. Predominantly conjugated to poly-SUMO2/3 rather than SUMO1 and the level of these conjugates undergo rhythmic variation, peaking at CT9-CT12. Sumoylation localizes it exclusively to the PML body and promotes its ubiquitination in the PML body, ubiquitin-dependent proteasomal degradation and the transcriptional activity of the CLOCK-ARNTL/BMAL1 heterodimer.
  • Cellular localization
    Nucleus. Cytoplasm. Nucleus, PML body. Shuttles between the nucleus and the cytoplasm and this nucleocytoplasmic shuttling is essential for the nuclear accumulation of CLOCK, target gene transcription and the degradation of the CLOCK-ARNTL/BMAL1 heterodimer. The sumoylated form localizes in the PML body. Sequestered to the cytoplasm in the presence of ID2.
  • Information by UniProt
  • Database links
  • Alternative names
    • ARNT like protein 1 brain and muscle antibody
    • Arntl antibody
    • Aryl hydrocarbon receptor nuclear translocator like antibody
    • Aryl hydrocarbon receptor nuclear translocator like protein 1 antibody
    • Aryl hydrocarbon receptor nuclear translocator-like protein 1 antibody
    • Basic helix loop helix PAS orphan MOP3 antibody
    • Basic helix loop helix PAS protein MOP3 antibody
    • Basic-helix-loop-helix-PAS protein MOP3 antibody
    • bHLH PAS protein JAP3 antibody
    • bHLH-PAS protein JAP3 antibody
    • bHLHe5 antibody
    • BMAL 1 antibody
    • BMAL1_HUMAN antibody
    • BMAL1c antibody
    • Brain and muscle ARNT like 1 antibody
    • Brain and muscle ARNT-like 1 antibody
    • CG8727 PA antibody
    • Class E basic helix-loop-helix protein 5 antibody
    • cycle antibody
    • JAP 3 antibody
    • JAP3 antibody
    • Member of PAS protein 3 antibody
    • Member of PAS superfamily 3 antibody
    • MGC47515 antibody
    • MOP 3 antibody
    • MOP3 antibody
    • PAS domain-containing protein 3 antibody
    • PASD 3 antibody
    • PASD3 antibody
    • TIC antibody
    see all

Images

  • All lanes : Anti-BMAL1 antibody - ChIP Grade (ab3350) at 1/500 dilution

    Lane 1 : U251 cell lysate
    Lane 2 : U87-MG cell lysate
    Lane 3 : NIH-3T3 cell lysate

    Lysates/proteins at 25 µg per lane.

    Predicted band size: 69.4 kDa
    Observed band size: 69 kDa
    why is the actual band size different from the predicted?

  • ICC/IF image of ab3350 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab3350, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Anti-BMAL1 antibody - ChIP Grade (ab3350) at 1 µg/ml + HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Developed using the ECL technique.

    Predicted band size: 69.4 kDa
    Observed band size: 75 kDa why is the actual band size different from the predicted?
    Additional bands at: 51 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 3 minutes


    The 75 kDa band observed is comparable to the molecular weight seen with other commercially available antibodies to BMAL1.

References

This product has been referenced in:
See all 35 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Mouse Cell (Hypothalamic Suprachiasmatic Nucleus)
Permeabilization
Yes - 0.5% Triton X-100
Specification
Hypothalamic Suprachiasmatic Nucleus
Blocking step
Serum as blocking agent for 24 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 4°C
Fixative
Paraformaldehyde

Kevin Zhang

Verified customer

Submitted May 16 2017

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (hepatocytes)
Permeabilization
Yes - : 0.1% Triton X-100 in TBS for 5-10 minutes
Specification
hepatocytes
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 23°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Dec 11 2015

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 22°C
Sample
Human Cell (Hepatocyte)
Specification
Hepatocyte
Permeabilization
No
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Mar 20 2014

Application
Western blot
Loading amount
50 µg
Gel Running Conditions
Non-reduced Denaturing (10%)
Sample
Mouse Cell lysate - whole cell (primary hepatoctyes)
Specification
primary hepatoctyes
Blocking step
I-Block(Applied biosystems) as blocking agent for 30 minute(s) · Concentration: 2µg/mL · Temperature: 22°C

Abcam user community

Verified customer

Submitted Jul 05 2013

Answer

Thank you for contacting us.

I could suggest buying ab150035.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Question

Product code: 3350
Lot number: GR49442-1
Inquiry: Dear sir/madam, Some time ago, I purchased the anti-BMAL1 antibody (ab3350) from your company. Initially, I was not able to see the predicted band using WB, but after some time of optimization, I seem to have succeeded in visualizing some (faint) bands a little lower than the 70 kD mark in murine heart tissue lysates. However, as of yet I have not seen the same band in the positive control suggested in the datasheet, i.e. HeLa whole cell lysate, which makes it difficult to be certain of the correct height of seen bands. Would it be possible for you to send me a positive control (protein or lysate), guaranteed to show a band, so I can ascertain myself of the correctness of these bands? If so, I would be very much obliged. For your information, I enclosed a picture of some blots, for which I used different conditions. Other conditions I have tried I will list below: * amount of protein loaded: 20 - 40 - 60 µg * blocking conditions: 20 min.@RT in 5% milk - 5% BSA * detection method: ECL (Amersham) * electrophoresis: 10% PAA gels (0.75 mm), ∼2.5 h at 120 V, samples boiled and loaded in sample buffer containing β-mercaptoethanol * transfer conditions: PVDF membranes, 1.5 h at 120 V in blotting buffer (2% methanol), cooled and stirred * primary antibody conditions: 1/500 - 1/1000 in 5% milk - 5% BSA, overnight@4°C * secondary antibody conditions: Swine anti-Rabbit 1/1000 in 5% milk - 5% BSA, 2 h@RT * wash steps: 3x15 min.@RT with TBS-Tween (after 1st AB and after 2nd AB) Many thanks in advance! Kind regards,

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Answer

Thank you for your email. I am sorry to hear that you have been experiencing problems with this antibody.

This antibody has been tested with Hela cells and is guaranteed for working in WB including other cell line lysates. We can certainly provide the Hela cell lysates however these will be general lysates i.e. not the one which we used to test this antibody. Let me know if you are OK to purchase these.

I have further read the details you have kindly provided and have following further questions for better understanding of the problem;

- How the antibody was stored?
- When the antibody was ordered? Could you provide the purchase order number?
- I agree the antibody does seem detecting very faint band at 70kDa. Could you provide details which lysis buffer was used and how the tissue lysates were prepared.

This protein has 5 isoforms (http://www.uniprot.org/uniprot/Q9WTL8) the antibody will detect all the isoforms if they are expressed. Please check literature about the published isofiorms in murine heart tissue.

Thank you very much for your cooperation. I will look forward to hearing from you soon.

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Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunofluorescence
Sample
Mouse Tissue sections (Brain Suprachiasmatic nuclei)
Specification
Brain Suprachiasmatic nuclei
Fixative
Paraformaldehyde
Antigen retrieval step
None
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2%

Abcam user community

Verified customer

Submitted Oct 10 2005

Answer

Thank you for your enquiry. We currently do not have an image of the western blot with ab3350, all the information, including images, we have for all our products is listed on the on-line datasheets for your convenience and we update these as soon as we get more data. Ab3350 was tested on hamster BMAL1/aryl hydrocarbon nuclear translocator 3 (ARNT3)fusion construct overexpressed in E. coli (a 110 kDa protein) as well as recombinant human BMAL1. The predicted MW of BMAL1 is 69.4 kDa. Please do not hesitate to contact us again if you need further assistance,

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