An equal quantity of protein in 16 μL was loaded per well of pre-cast mini-protean TGX gels 4–20% gels (Biorad), with one gel per timeseries. Electrophoresis was applied according to the manufacturers protocol with a tris–glycine-SDS running buffer (Biorad). Protein transfer onto nitrocellulose membranes (Biorad) was performed using the trans-blot turbo mini kit (Biorad). The membranes were allowed to dry at room temp for 1 h prior to quantifying the total protein using the Li-Cor total protein stain protocol and Odyssey blot-imager (Li-Cor). The membranes were blocked using 5% non-fat milk powder (Marvel) in Tris-buffered saline (TBS) for 1 h at room temperature before incubating overnight at 4 °C with primary antibody (rabbit anti-bmal-1, abcam ab93806) diluted 1:3000 in 2.5% milk in TBS + 0.1% tween-20. After washing, membranes were incubated for 1 h at room temp with secondary antibody, donkey anti-rabbit IRDye 800CW (Licor, 925–32,213) diluted 1:10,000 in TBS + 0.2% tween-20. The membranes were washed extensively before imaging with the Li-Cor Odyssey blot-imager. The 800 nm fluorescence signal from BMAL-1 was quantified using the Image Studio Lite software (Li-Cor Bioscience) and normalised to total protein. Representative Western blot images are presented in Fig. S1 in the supplementary material.
Ab93806
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab93806 is a laboratory product offered by Abcam. It is a primary antibody designed for use in various experimental techniques. The core function of this product is to serve as a detection reagent for the specific target it is intended to recognize.
Automatically generated - may contain errors
Lab products found in correlation
Ab6276, by Abcam (2 mentions)
Ab5089, by Abcam (2 mentions)
Ab181602, by Abcam (2 mentions)
Odyssey blot imager, by LI COR (1 mentions)
Irdye 800cw donkey anti rabbit, by LI COR (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Tris glycine sds running buffer, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Ab7030, by Abcam (1 mentions)
Ab124964, by Abcam (1 mentions)
Bovine serum albumin (bsa), by neoFroxx (1 mentions)
Ab225844, by Abcam (1 mentions)
Pierce bca kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Mini trans blot cell system, by Bio-Rad (1 mentions)
Gapdh 2118, by Cell Signaling Technology (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Las 4000 imaging system, by Fujifilm (1 mentions)
Glucosamine, by Beyotime (1 mentions)
Glucose oxidase assay kit, by Applygen (1 mentions)
21 protocols using ab93806
1
Western Blot Analysis of BMAL-1 Protein
2
Muscle Transcription Factor Regulation Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In vivo skeletal muscle ChIP was performed with sonicated nuclear extract prepared from formaldehyde-cross-linked gastrocnemius muscle according to [137 (link)]. For immunoprecipitation, we used anti-BMAL1 (ab93806, Abcam), anti-REV-ERBα (generous gift from Ron Evans), anti-RNA Polymerase II (#MMS-126R; clone 8WG1, Biolegends), anti-NCOR1 (#20018-1-AP, Protein Tech), anti-HDAC3 (ab7030, Abcam), or rabbit IgG (2027x, Santa Cruz). DNA was column purified and used for sequencing or real-time qPCR (enrichment expressed as fold-change relative to IgG; primer sequences used are listed in S4 Table ).
3
Cardiac Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cardiac tissues and cells were lysed with RIPA lysis buffer containing the protease inhibitor. Protein concentrations were determined by the Pierce BCA kit (Thermo scientific). Equal amounts of protein were loaded and separated by 10% SDS‐PAGE, and then transferred onto nitrocellulose filter membranes for blotting. Membranes were briefly washed and blocked in 5% Bovine Serum Albumin (BioFroxx) for 1 hour and then incubated with primary antibody solution overnight at 4 ℃. The following antibodies were used: GAPDH (Proteintech, 10494‐1‐AP) with 1:5000 dilution, Bmal1 (Abcam, ab93806) with 1:2000 dilution, α‐SMA (Abcam, ab124964) with 1:10000 dilution, Fibronectin (abcam, ab2413) with 1:5000 dilution, collagen I (Proteintech, 14695‐1‐AP) with 1:5000 dilution, AKT (T‐AKT) (CST, 4691) with 1:2000 dilution, phosphorylated AKT (P‐AKT) (CST, 4060) with 1:2000 dilution, ANP (atrial natriuretic peptide; abcam, ab225844) with 1:2000 dilution. Membranes were incubated with either goat‐rabbit or anti‐mouse horse radish peroxidase‐conjugated secondary antibody (Proteintech) for 1 hour. Finally, the membranes were transferred to the Super Lumia ECL Plus HRP Substrate Reagent (advansta), and images were collected using Chemiluminescent Imaging System (Tanon, China). Quantification analysis was performed using Image J software.
4
Western Blot Analysis of Bladder Urothelium
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell lysates from bladder urothelium and cultured cells were lysed with radioimmunoprecipitation assay (RIPA) buffer containing proteinase inhibitors, which were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA) using a Mini Trans-Blot Cell system (Bio-Rad Laboratories). Membranes were blocked with 5% bovine serum albumin diluted in TBST (BSA/TBST) and incubated with primary antibodies diluted in 1% BSA/TBST followed by incubation with horseradish peroxidase-conjugated secondary antibodies diluted in 1% BSA/TBST and developed for reading by enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Thermo). Images were acquired with the LAS-4000 imaging system (Fujifilm Life Science, Tokyo, Japan). Anti-Cx43 (C6219, Sigma-Aldrich, 1:8000), anti-BMAL1 (ab93806, Abcam, 1:500), anti-beta actin (ab6276, Abcam, 1:5000) and anti-GAPDH (GAPDH, 2118, Cell Signaling Technology, Danvers, MA, USA, 1:5000) were used as the primary antibodies.
5
Berberine and Oxidative Stress Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Berberine (B3251) (purity > 98%) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Glucosamine (purity > 99%) was purchased from the Beyotime Biotechnology Company (Nanjing, China). The Roswell Park Memorial Institute 1640 medium (RPMI-1640) growth medium was purchased from (Hyclone Co., Logan, UT, USA). Fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Lonza Walkerrsville (GIBCO CO., Allendale, NJ, USA). The concentrations of ROS and H2O2 in the liver were measured using a mouse ROS ELISA kit (RD-RX20367-48T) and H2O2 ELISA kit (RD-RX21392-48T) (Beijing Ruida Henghui Technology Development Co., Ltd., Beijing, China). HepG2 cells were purchased from the American Type Culture Collection (ATCC). Antibodies against Clock (ab93804) and Bmal1 (ab93806) were purchased from Abcam (Abcam, Cambridge, MA, USA). The cell counting kit-8 (CCK8) assay kit was purchased from DoJinDo (DoJinDo ChemTech Lim, Kumamoto, Tokyo, Japan). The Glucose Oxidase Assay Kit was obtained from APPLYGEN (APPLYGEN, Beijing, China). The cellular ROS and H2O2 assay kit was purchased from Beyotime (Biotechnology, Nanjing, China). Machine-filtered, pure water (Milli-Q) was used.
6
Bmal1 Chromatin Immunoprecipitation in C2C12 Myotubes
7
Circadian Rhythm Protein Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein concentrations were measured by the bicinchoninic acid assay (CW0014, CWBIO Company) after the total protein extracts were collected from pinealocytes using the radioimmunoprecipitation lysis buffer (CW2333, CWBIO Company). Equal amounts of protein from each sample were separated using SDS-PAGE and transferred onto PVDF membranes using electroblotting. The membranes were incubated in a solution containing 5% skim milk in phosphate buffer saline at room temperature for 1 h. The membranes were incubated with a CLOCK rabbit polyclonal antibody (1:1000, ab461, Abcam) and BMAL1 rabbit polyclonal antibody (1:1000, ab93806, Abcam) overnight at 4°C. After washing the membranes in Tris-buffered saline-Tween, they were incubated with goat anti-rabbit horseradish peroxidase-conjugated immunoglobulin (Ig) G (1:8000, CW0103, CWBIO Company) for 1 h at room temperature. The blot bands were detected using the Ecl Western blot kit (1627003, Millipore) after washing. The intensity of the signals for CLOCK and BMAL1 were quantified using Image-Pro plus software and were normalised relative to the values obtained for β-ACTIN (1:4000, CW0096, CWBIO Company).
8
Western Blot Analysis of Circadian Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed using RIPA buffer (20 mM Tris, pH 7.5, 2 mM EDTA, 150 mM NaCl, 1% NP40, and 1% sodium deoxycholate), supplemented with a protease inhibitor cocktail (Roche Complete), Laemmli sample buffer was added and samples incubated at 95°C for 5 min. Proteins were separated on a 10% polyacrylamide gel and transferred to polyvinylidene difluoride membrane (Amershan Hybond P PVDF membrane, Merck). Membranes were blocked in 5% milk in PBS/0.1% Tween-20, followed by incubation with anti-BMAL1 (Ab93806, abcam), anti-RORC (AFKJS-9, eBioscience) or anti-β-actin (A5441, Sigma) primary antibodies and appropriate HRP-conjugated secondary antibodies (DAKO). A chemiluminescence substrate (West Dura, 34076, Thermo Fisher) was used to visualise proteins using a ChemiDoc XRS+ imaging system (BioRad).
9
Western Blot Analysis of BMAL1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from cells using RIPA buffer and quantified using a BCA Protein assay kit (Pierce, Rockford, IL) following the manufacturer’s protocol. Sample were then mixed with SDS loading buffer, electrophoresed on 10% (vol/vol) acrylamide gels, transferred onto PVDF membranes, and blocked with 5% milk for 1 h before incubation with a primary antibody. The antibodies used for western blotting were rabbit anti-BMAL1 (ab93806, Abcam, Cambridge, UK) and anti-β-tubulin (clone 10G10, Wako, Osaka, Japan). The blots were then developed using ECL Prime detection reagent (GE, Waukesha, WI, USA). Raw images are in Fig. S8 .
10
Quantification of Smad3 Phosphorylation in Brown Preadipocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
40–50 μg of total protein from tissues or cell homogenates were used for each sample on SDS-PAGE gel. After electrophoresis, protein were transferred to PVDF membrane, blotted using specific primary and secondary antibody and detected by chemiluminescence (Supersignal; Pierce Biotechnology), as previously described49 (link). Smad3 phosphorylation in immortalized primary brown preadipocytes was assessed at 1 hour after indicated ligand treatment. The primary antibodies used were: Rev-erbα, PA5-29865 (Thermo Scientific), Bmal1, AB93806 (Abcam), UCP-1, AB3038 (Millipore); CEBPβ, sc-150, TBP, sc-204 (Santa Cruz); Smad3, 04-1035, phosphor-Smad3 (Ser 423/425), 07-1389 (Millipore).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!