The TPBC and TNBC sections were hydrated and treated with 1.5% hydrogen peroxide for 1 h at 37°C, followed by treatment in 0.05 M citrate-buffered saline (pH 6.0) at 95°C for antigen retrieval. The TPBC and TNBC sections were then incubated in 5% goat serum (Gibco) for 1 h at 37°C prior to incubation with the primary antibodies (1:200 dilutions) anti-BMAL1, anti-PER1, anti-ASMT (Abcam), anti-CLOCK (Abcam, 1:1000), and anti-serotonin N-acetyltransferase N-terminal (Sigma-Aldrich, 1:200) for 1 h at 37°C and overnight at 4°C. Subsequently, the TPBC and TNBC sections were incubated with the biotinylated goat anti-rabbit IgG (Santa Cruze Biotechnology, 1:200) for 1 h at 37°C, followed by incubation with avidin-biotin peroxidase complex (1:200) and dehydration with ethanol. Data are presented as the percentages of positive areas of ASMT, NAT, CLOCK, BMAL1, and PER1 relative to the total area. Six uncontinuous fields were selected from one slide at a magnification of 200×.
Anti clock
Manufactured by Abcam
Sourced in United Kingdom, United States, Israel
Anti-CLOCK is a primary antibody that specifically targets the CLOCK protein. CLOCK is a key regulator of circadian rhythms, playing a central role in the body's internal clock. This antibody can be used to detect and study the CLOCK protein in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti bmal1, by Abcam (4 mentions)
Anti per1, by Abcam (3 mentions)
Anti per2, by Abcam (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Anti serotonin n acetyltransferase n terminal, by Merck Group (1 mentions)
Complete mini edta free, by Roche (1 mentions)
Ecl plus chemiluminescence, by GE Healthcare (1 mentions)
Protein assay dye reagent concentrate, by Bio-Rad (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Anti eaac1, by Abcam (1 mentions)
Ecl prime hrp detection kit, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Anti β actin, by Beyotime (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Sds page gel, by Beyotime (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
7 protocols using anti clock
1
Circadian Clock Protein Expression in TPBC and TNBC
2
Western Blot Analysis of Circadian Clock Proteins
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Whole-cell protein extracts were prepared by lysing cells in RIPA buffer containing protease inhibitors (cOmplete, Mini, EDTA-free; Roche, Welwyn Garden City, United Kingdom). Protein yield was quantified using the Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Hemel Hempstead, United Kingdom). Equal amounts of protein were separated by SDS-PAGE before wet transfer onto PVDF membrane (GE Healthcare, Buckinghamshire, United Kingdom). Nonspecific binding sites were blocked by overnight incubation with 5% nonfat dry milk in Tris-buffered saline with 1% Tween 20 [130 mM NaCl, 20 mM Tris (pH 7.6), and 1% Tween 20]. The following primary antibodies were purchased from Abcam (Cambridge, United Kingdom): anti-CLOCK (catalog number ab3517, diluted 1:3000); anti-BMAL1 (ab3350, 1:375); anti-CRY1 (ab54649, 1:500); anti-CRY2 (ab38872, 1:2000); anti-PER1 (ab3443, 1:300); anti-PER2 (ab179813, 1:300); and anti-β-actin (ab8226, 1:10,000). Protein complexes were visualized with ECL Plus chemiluminescence (GE Healthcare). The Western blots are collated in Supplemental Fig. 1 .
3
Western Blot Analysis of Circadian Proteins
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The amount of protein was determined using the BCA protein assay (Thermo Scientific, Rockford, IL, USA), and the same amounts of proteins were normalized for total protein. The protein samples were boiled in RIPA buffer (20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS) and protease inhibitor cocktail; Sigma-Aldrich, St Louis, MO, USA), separated by SDS–polyacrylamide gel electrophoresis (PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Non-specific binding was blocked with 5% skim milk in PBS-Tween20, and proteins were probed with anti-EAAC1 (Abcam, Cambridge, MA) at 1:1,000 dilution, anti-PER1 (Abcam) at 1:200, anti-PER2 (Abcam) at 1:1,000, anti-BMAL1 (Abcam) at 1:1,000, anti-CLOCK (Abcam) at 1:1,000 and anti-β-actin (Sigma-Aldrich) at 1:10,000 dilution. After a wash with PBS-Tween20, the horseradish peroxidase-labelled secondary antibodies were probed and detected with the ECL prime HRP detection kit (GE Healthcare, Piscataway, NJ, USA). We performed the quantification of the EAAC1 level using a serial dilution of ZT14 samples as the standard curve.
4
Antibody Validation for Circadian Clock Studies
5
Western Blot Analysis of Clock Proteins
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NK cells from control or chronic shift‐lag mice were transfected with Per1‐siRNA and Per2‐siRNA or negative control siRNA. After 24 hours, the cells were pelleted and lysed in RIPA buffer (Beyotime). Total protein was isolated, and the concentration was estimated using a BCA assay (Beyotime). Equal amounts of protein (50 μg) were separated on 12% SDS‐PAGE gels (Beyotime) and transferred to PVDF membrane (Thermo Fisher). The membranes were blocked with 5% BSA for 2 hours at room temperature and subsequently incubated overnight at 4°C with the following specific antibodies: anti‐Per1 (1:1000, Abcam), anti‐Per2 (1:1000, Abcam), anti‐CLOCK (1:1000, Abcam) and anti‐β‐actin (1:1000, Beyotime). The following day, the membranes were washed and incubated with a secondary antibody (rabbit, 1:1000, Beyotime) for 2 hours at room temperature. Protein bands were visualized using ECL Western blotting substrate on a BioRad ChemiDoc MP system (BioRad, CA).
6
Quantifying Metabolic Regulators in Cells
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Physcion was purchased from MedChemExpress. Horse serum was purchased from GIBCO Company. Anti-SREBP-1, anti-PPARα, anti-CLOCK, and anti-BMAL1 antibodies were purchased from Abcam (Cambridge, MA, United States). Anti-AMPKα, anti-phospho-AMPKα, and anti-cleaved caspase-1 antibodies were purchased from Cell Signaling Technology (Beverly, MA, United States). Anti-P2X7R, anti-IL1β, and anti-caspase1 antibodies were purchased from Santa Cruz Biotechnology. The BCA Protein Assay Kit was purchased from Solarbio (Beijing, China).
7
Circadian Regulation of Cardiac Proteins
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Samples were prepared and collected every 3 hours starting from ZT0, in a similar fashion as previous studies (Wang et al. 2012) . Briefly, left ventricle of the hearts were homogenized in RIPA buffer. The samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to PVDF membranes. The primary antibodies used in this study were anti-Cav1.2 (Alomone, Jerusalem, Israel) , anti-CLOCK (Abcam), anti-Akt (total Akt) (Cell Signal Technology), anti-pAkt T308(Cell Signal Technology), anti-pAkt S473(Cell Signal Technology), anti-PDK1(Cell Signal Technology) and anti-βtubulin(Cell Signal Technology). βtubulin was used as the internal reference. All measurements were repeated for at least 3 times.
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