BMAL1 antibody | knockout validation | Abcam ab93806

This is a knockout-validated antibody summary, based on the publication "Astrocyte deletion of Bmal1 alters daily locomotor activity and cognitive functions via GABA signalling", as cited below [1]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

Antibody information

Rabbit polyclonal IgG

Company: Abcam

Antibody: BMAL1

Catalog number: ab93806

Summary: Rabbit polyclonal IgG against a synthetic peptide, corresponding to a region within amino acids 575-625 of human BMAL1. Reacts with human and mouse. It is predicted to work with rat, sheep, rabbit, horse, chicken, guinea pig, cow, dog, pig, Xenopus, zebrafish, and primates.

Suitable for western blot, immunocytochemistry/immunofluorescence and immunoprecipitation.

Validation Method

Western blot | Immunohistochemistry

Sample

Brain from control and Bmal1-cKO mice. WB: Cortices were homogenized in lysis buffer with the following final concentrations: 50 mM Tris-HCl pH 7.5, 250 mM sucrose, 5 mM sodium pyrophosphate (NaPPi), 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, 50 mg ml 1 leupeptin and 50 mg ml 1 soybean trypsin inhibitor. Following lysis, SDS was added to a final of 0.2%. IHC: Brains were post-fixed overnight in 4% paraformaldehyde in PBS and 30 mm slices were prepared Cryostat (Leica).

Blocking agent

IHC: 10% goat serum in PBS.

Primary incubation

WB: 1:1,000 dilution.

IHC: 1:200 dilution at 4 C overnight.

Secondary incubation

WB: 1:5,000 dilution horseradish peroxidase-coupled secondary antibodies (Abcam, ab6721).

IHC: 1:1,000 dilution goat anti-rabbit Alexa-488 or Alexa-546 secondary antibodies (Invitrogen, A-11034, and A-11010) for 2 h.

Detection

WB: westernlight chemiluminescence detection system (ECL, GE Healthcare Bio-Sciences AB), exposed and photographed in an ImageQuant LAS 4,000 mini (GE Healthcare Bio-Sciences AB).

IHC: inverted laser scanning confocal microscope (TCS SP5 microscope using a 20 or 40 objective (Leica Microsystems)).

Figure

Glast:CreERT2-driven Td-TOMATO-positive SCN astrocytes express BMALl1. Representative micrographs of BMAL1 immunostaining in the SCN of control or Bmal1cKO-Td-Tomato animals in 12:12 h LD cycles (DAPI in blue, TOMATO in red and BMAL1 in green). Scale bars, 50 μm and 20 μm in the higher magnification images. Representative images of cortical BMAL1 western blotting in Bmal1cKO as compared with control mice (n = 3 animals per group and time point). Please see Figure 1b and 3c in the article [1].

References
  1. Barca-Mayo O, Pons-Espinal M, Follert P, Armirotti A, Berdondini L, De Pietri Tonelli D. Astrocyte deletion of Bmal1 alters daily locomotor activity and cognitive functions via GABA signalling. Nat Commun. 2017;8:14336 pubmed publisher