Bmal1

Manufactured by Fortis Life Sciences

Sourced in United States

BMAL1 is a protein that plays a key role in the regulation of circadian rhythms, the 24-hour cycles of physiological processes in living organisms. It functions as a transcription factor, activating the expression of genes involved in the circadian clock. BMAL1 is an essential component of the molecular machinery that controls the body's internal time-keeping system.

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Lab products found in correlation

Chemidoc xrs imager, by Bio-Rad (3 mentions) Supersignal west femto, by Thermo Fisher Scientific (3 mentions) P h2ax, by Cell Signaling Technology (2 mentions) Clarity western ecl chemiluminescent, by Bio-Rad (2 mentions) Nr1d1, by Cell Signaling Technology (1 mentions) Image lab software, by Bio-Rad (1 mentions) Protease inhibitor, by Thermo Fisher Scientific (1 mentions) Clarity western ecl, by Bio-Rad (1 mentions) β tubulin, by Cell Signaling Technology (1 mentions) Gapdh, by Santa Cruz Biotechnology (1 mentions) Horseradish peroxidase conjugated secondary antibody, by Southern Biotech (1 mentions) Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions) Amersham imager 600, by Cytiva (1 mentions) Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Rev erbα, by Cell Signaling Technology (1 mentions) C caspase 3, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

Anti-BMAL1 Bmal1 antibody (A302-616A )

5 protocols using bmal1

Nuclear Fractionation and Immunoblotting of Circadian Proteins

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Antibody for detecting mouse CRY1 was provided by the laboratory of Aziz Sancar and was described previously (Ye et al., 2011 (link)). Antibodies for detection CLOCK (Bethyl Laboratories), BMAL1 (Bethyl Laboratories), CRY2 (Bethyl Laboratories), NR1D1 (Cell Signaling Technology), and NR1D2 (Santa Cruz Biotechnology) were from commercial sources.
For nuclear fractionation, cells were detached from the plate by trypsinization. After PBS wash, cells were lysed with hypotonic buffer (10 mM HEPES pH7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.3% Igepal-CA630). After centrifugation (400 g for 5 min), the supernatants were collected as cytosol fractions. The nuclear pellets were washed with hypotonic buffer and were lysed with NUN buffer (20 mM HEPES pH7.4, 300 mM NaCl, 1 M urea, and 10% glycerol). After centrifugation (15,000 g for 15 min), the supernatants were collected as the enriched nuclear fractions.
The protein concentrations of lysates were estimated by the QuantiChrom^TM Total Protein Assay Kit (BioAssay System). Equal amounts of total proteins were applied to the Western blot experiments.
The Western blot results were quantified by the Image Lab software (Bio-Rad Laboratories). For estimating the relative levels of CRY1-CLOCK-BMAL1 in the nucleus, CRY1 levels in the nuclear fractions were first normalized to the BMAL1 levels and then compared to the group without 4-OHT treatment.

Chiou Y.Y., Li T.Y., Yang Y, & Sancar A. (2020). A Sextuple Knockout Cell Line System to Study the Differential Roles of CRY, PER, and NR1D in the Transcription-Translation Feedback Loop of the Circadian Clock. Frontiers in Neuroscience, 14, 616802.

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Protein Extraction and Immunoblotting Protocol

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Frozen skin tissue samples were homogenized in liquid nitrogen using a mortar and pestle. Protein lysate was extracted from tissues using 1x RIPA lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, and 1% sodium deoxycholate) plus 1x protease inhibitors (Fisher Scientific; Cat # 88666), and from cells using 1x lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Tween-20) plus protease inhibitors and to determine the protein levels, the immunoblotting protocol was followed as previously described by us (Gaddameedhi et al., 2011 (link); Gaddameedhi et al., 2015 (link)). The following antibodies were used: GAPDH (Santa Cruz Biotechnology; Cat # sc-25778), BMAL1 (Bethyl Laboratories; Cat # A302–616A), β-Tubulin and pH2A.X (Cell Signaling Technology; Cat #s 86298T and 9718S, respectively). The appropriate HRP-conjugated secondary antibody was used for detection with chemiluminescence (Clarity Western ECL, Bio-Rad, and/or SuperSignal West Femto, Thermo Fisher Scientific) using a Bio-Rad ChemiDoc XRS+ imager.

Kaplan D., Ferrari I., Bergami P.L., Mahler E., Levitus G., Chiale P., Hoebeke J., Van Regenmortel M.H, & Levin M.J. (1997). Antibodies to ribosomal P proteins of Trypanosoma cruzi in Chagas disease possess functional autoreactivity with heart tissue and differ from anti-P autoantibodies in lupus. Proceedings of the National Academy of Sciences of the United States of America, 94(19), 10301-10306.

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Protein Expression Analysis of Frozen Kidney Tissue

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Frozen kidney tissue samples were homogenized in liquid nitrogen using a mortar and pestle, protein lysate was extracted using RIPA lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Nonidet P-40, and 1% sodium deoxycholate), and conventional immunoblotting procedures were used to determine the levels of selected proteins involved in DNA repair, proliferation, and the circadian clock. The following antibodies were used: Actin and XPA (Santa Cruz Biotechnology, cat. #s sc-1616 and sc-28353 respectively), BMAL1 (Bethyl Laboratories, cat. # A302-616A), and γH2AX (Ser139), RPA32/RPA2 and PCNA (Cell Signaling Technology, cat. #s 9718S, 2208, and 2568S respectively). The appropriate anti-mouse, anti-rabbit, anti-rat, and anti-goat HRP-conjugated secondary antibody was used for detection with Clarity Western ECL chemiluminescent (Bio-Rad Laboratories) and/or SuperSignal West Femto (Thermo Fisher Scientific) reagent method with Bio-Rad ChemiDoc XRS+ imager.

Dakup P.P., Porter K.I., Little A.A., Gajula R.P., Zhang H., Skornyakov E., Kemp M.G., Van Dongen H.P, & Gaddameedhi S. (2018). The circadian clock regulates cisplatin-induced toxicity and tumor regression in melanoma mouse and human models. Oncotarget, 9(18), 14524-14538.

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Western Blotting Protocol for Protein Analysis

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Western blotting was performed as published previously^{58 (link)}. Tissue was lysed via sonication in M-PER with protease and phosphatase inhibitors (10 μl/ml, Halt Protease and Phosphatase Inhibitor Cocktail, Thermo Scientific Inc, Rockford, IL, USA) and centrifuged to clear. Supernatant concentrations were detected by BCA Assay (Pierce) and adjusted. Protein was resolved on 9% Tris–glycine polyacrylamide gels under reducing conditions and transferred to nitrocellulose membrane (Bio-rad, Hercules CA). Membranes were blocked for 45 min in 5% milk in tris buffered saline containing 0.1% tween-20 solution (Boston Bioproducts, Ashland, MA). Membranes were incubated overnight at 4 °C in specific antibodies to pERK p44/42, GSK3β, Arg3.1(Arc) (1:1000, Cell Signaling Technology, MA, USA) PER1 (1:1000 Invitrogen, CA, USA), PER2 (1:250 Invitrogen), or BMAL1 (1:1000 Bethyl Laboratories, TX, USA) or for 2 h at RT in antibody specific to β -actin(Sigma-Aldritch, MO, USA) in tris buffered saline with 0.1% tween20 (TBS-T) with 2% milk, washed 3 × 10 min in TBS-T, and incubated for 1 h at room temperature in horseradish peroxidase-conjugated secondary antibody (1:7500, SouthernBiotech, AL, USA), in TBS-T with 2% milk. After washing with TBS-T, bands were visualized with enhanced chemiluminescence (ECL; Thermo scientific) using an image analyzer (Amersham imager 600).

Mahoney H., Peterson E., Justin H., Gonzalez D., Cardona C., Stevanovic K., Faulkner J., Yunus A., Portugues A., Henriksen A., Burns C., McNeill C., Gamsby J, & Gulick D. (2021). Inhibition of casein kinase 1 δ/ε improves cognitive performance in adult C57BL/6J mice. Scientific Reports, 11, 4746.

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Protein Analysis of Heart Tissue Samples

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Frozen heart tissue samples were homogenized in liquid nitrogen using a mortar and pestle. Protein lysate was extracted from tissues using 1X RIPA lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, and 1% sodium deoxycholate), and from cells using 1X lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Tween-20). Conventional immunoblotting procedures were used to determine the levels of selected proteins involved in DDR signaling and the circadian clock. The following antibodies were used: Actin (Santa Cruz Biotechnology; Cat #s sc-1616), Bmal1 (Bethyl Laboratories; Cat #s A302-616A), c-Caspase 3, pH2a.x, H2a, and Rev-erbα (Cell Signaling Technology; Cat #s 9664P, 9718S, 12349S and 13418S respectively). The appropriate anti-mouse, anti-rabbit, and anti-goat HRP-conjugated secondary antibodies (Sigma-Aldrich) were used for detection with Clarity Western ECL chemiluminescent (Bio-Rad Laboratories) and/or SuperSignal West Femto (Thermo Fisher Scientific) reagent methods with Bio-Rad ChemiDoc XRS+ imager.

Dakup P.P., Porter K.I., Gajula R.P., Goel P.N., Cheng Z, & Gaddameedhi S. (2020). The circadian clock protects against ionizing radiation-induced cardiotoxicity. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(2), 3347-3358.

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