Baicalin and 3-MA were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). BAPTA-AM was purchased from Selleck Chemicals (Shanghai, China). Anti-Bax, anti-Bcl-xl, anti-cleaved caspase 3, anti-PARP, anti-mTOR, anti-p-mTOR, anti-AKT, anti-p-AKT, anti-cyclin D1, anti-cyclin B1, anti-cyclin A, anti-ZO-1, anti-Catenin, anti-Vimentin, and anti-β-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-LC3B, anti-MMP-2, and anti-beclin 1 antibodies were purchased from Novus Biologicals (Littleton, CO, USA). Anti-HMGB1 and anti-p62/SQSTM1 antibodies were purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase (HRP)-conjugated and FITC-conjugated antirabbit or antimouse IgG antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
Anti cyclin a
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-Cyclin A is a laboratory reagent used to detect and quantify the Cyclin A protein. Cyclin A is a key regulator of cell cycle progression, playing a role in the transition from G1 to S phase and the S to G2 phase. The Anti-Cyclin A reagent can be used in various experimental techniques, such as immunoblotting and immunohistochemistry, to analyze the expression and localization of Cyclin A in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti bax, by Cell Signaling Technology (6 mentions)
Anti cyclin d1, by Cell Signaling Technology (6 mentions)
Anti cyclin b1, by Cell Signaling Technology (6 mentions)
Anti p21, by Cell Signaling Technology (5 mentions)
Anti bcl 2, by Cell Signaling Technology (4 mentions)
Anti cdk2, by Cell Signaling Technology (4 mentions)
Anti akt, by Cell Signaling Technology (3 mentions)
Anti p akt, by Cell Signaling Technology (3 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Anti cyclin e, by Cell Signaling Technology (3 mentions)
Anti p21 waf1 cip1, by Cell Signaling Technology (3 mentions)
Anti caspase 3, by Cell Signaling Technology (3 mentions)
Anti p53, by Cell Signaling Technology (3 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Anti parp, by Cell Signaling Technology (2 mentions)
Anti p27, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti e2f1, by Cell Signaling Technology (2 mentions)
Anti cdk4, by Cell Signaling Technology (2 mentions)
Anti skp2, by Cell Signaling Technology (2 mentions)
19 protocols using anti cyclin a
1
Baicalin and 3-MA Modulate Autophagy and Apoptosis
2
Immunoblotting for Cell Cycle Regulators
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Membranes were incubated with antibodies diluted in PBS supplemented with 5% milk protein and incubated, with agitation, overnight at 4°C. Membranes were subjected to 3 × 5 min washes in PBS and incubated in appropriate secondary antibodies for 1 h at room temperature. The antibodies used for immunoblotting were as follows: anti-CDK18 (Santa Cruz Biotechnology: sc-176), anti-pCDK substrate (phosphor-[K/H]pSP) MultiMab rabbit Ab mix (Cell Signalling), anti-Cyclin A, anti-Cyclin E and anti-Cyclin B1 (all from Cell Signaling), anti-Histone H3 pSer10 (27 (link)), anti-CHK1 (Sigma Aldrich), anti-CHK1 pS317 (Cell Signaling), anti-Myc 9B11 clone (Cell Signalling), anti-RAD9 (Abcam and Bethyl laboratories), anti-RPA2 (Calbiochem), anti-RPA2 pT21 (Abcam), anti-RPA2 pS4/8 (Bethyl Laboratories), anti-KAP1 (Bethyl Laboratories), anti-KAP1 pS824 (Bethyl Laboratories), anti-ATRIP (Bethyl Laboratories), anti-ATR (Santa Cruz Biotechnology), anti-Actin (Abcam), anti-ORC2 (Bethyl Laboratories), anti-RAD17 (Bethyl Laboratories), RAD17 (Santa Cruz Biotechnology), anti-BrdU (AbD Serotec and BD), anti-RRM2 (Santa Cruz Biotechnology), HRP-secondary antibodies (DAKO) and Alexa-Fluor antibodies (Invitrogen).
3
Protein Expression Analysis Protocol
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Cells were lysed in protein extraction buffer (50 mM HEPES, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 mM Na2P2O7, 1 mM Na3VO4, 5 μg/mL aprotinin, 5 μg/mL leupeptin, 1 mM PMSF, and protease inhibitor cocktail). Lysates containing equal amounts of proteins were separated by SDS–PAGE and transferred onto polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The blots were blocked with a 5% skim milk solution and incubated with the following antibodies: anti-Dicer, anti-cyclin A, anti-cyclin D1, anti-cyclin E, anti-p21WAF1/CIP1, anti-EZH2,(Cell Signaling Technology, Danvers, MA), anti-HDAC2, anti-GAPDH and anti-CDK2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-N-cadherin, anti-E-cadherin, anti-vimentin and anti-fibronectin (BD Transduction, San Jose, CA), anti-DNMT1 (Abcam, Cambridge, MA) and anti-H3K27me3 (Millipore, Billerica, MA). The Immobilon™ Western blot detection system (Millipore, Billerica MA) was used to detect bound antibodies. The intensities of the Western blot bands were quantified using LAS 3000 (Fuji Photo Film Co., Japan).
4
Protein Expression Analysis by Western Blot
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Harvested cells were washed twice with phosphate-buffered saline (PBS), and proteins were extracted using 1× loading lysis buffer. Equal amounts of protein, as measured by the BCA protein assay, were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and transferred on to a polyvinylidenedifluoride (PVDF) membrane (Immobilon P, Millipore). The membrane was sealed in 5% skim milk for 1 h, and then incubated with primary antibody for 2 h. After washing with TBST for three times, it was incubated with horseradish peroxidase-bound secondary antibody for 2 h. Anti-PARP, anti-Caspase-3, anti-Caspase-9, anti-cyto C, anti-Bcl-2, anti-Bax, anti-PI3K, anti-p-PI3K, anti-Akt, anti-p-Akt, anti-CDK1, anti-Cyclin B1, anti-Cyclin A, anti-p21, anti-p27, anti-GAPDH, HRP-conjugated goat anti-rabbit IgG, and HRP-conjugated rabbit anti-mouse IgG antibodies were purchased from Cell Signaling Technology. After cleaning with TBST, the Universal ECL immunoblotting chemiluminescence solution (Vazyme, China) was used to display the bands, and relative expression of protein was analyzed by ImageJ.
5
Galectin-3 and Rb Protein Interactions
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Western blotting was performed using anti-galectin-3, anti-Rb, anti-phospho-Rb (Ser 807/811) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-SKP2, anti-p27KIP1, anti-p21WAF1/CIP1, anti-p15INK4B, anti-cyclin A, anti-cyclin D1, anti-CDK4, anti-E2F1, anti-GAPDH, and anti-lamin A/C antibodies (Cell Signaling, Danvers, MA, USA). The normalization control was anti-β-actin (Sigma, St. Louis, MO, USA). Immunoprecipitation was carried out with A/G agarose beads coated with anti-galectin-3 and anti-Rb (Santa Cruz Biotechnology) antibodies. The proteins were detected by western blot analysis using antibodies against anti-galectin-3, anti-Rb, and anti-E2F1. Mouse/rabbit IgG (Santa Cruz Biotechnology) was used as a negative control.41 (link)
6
Protein Expression Analysis Using Western Blot
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Cell lysates were prepared by using RIPA lysis buffer (Beyotime, China), and the total protein concentration was determined by using a BCA protein detection kit (Pierce Biotechnology). Target proteins were separated by 8-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% bovine serum albumin for 1 h, the PVDF membranes were incubated with primary antibodies at 4°C overnight. The following antibodies were used: anti-EVI1 (album, ab124934); anti-MECOM (ATLAS ANTIBODIES, HPA046537); anti-Lamin B (Beyotime, AF1408); anti-GAPDH (Beyotime, AF1186); anti-Akt (Cell Signaling Technology, 4691); anti-p-Akt (Cell Signaling Technology, 4060); anti-Cyclin A (Cell Signaling Technology, 4656); anti-p21 (Cell Signaling Technology, 2947); anti-CDK2 (Cell Signaling Technology, 2546); anti-Bcl-2 (Cell Signaling Technology, 4223); anti-Bax (Cell Signaling Technology, 5023); anti-Caspase 3 (Cell Signaling Technology, 9665); and anti-PTEN (Cell Signaling Technology, 9188). After washing with TBST, the membranes were incubated with secondary antibodies for 1 h. Signals were detected with an ECL detection reagent (Beyotime).
7
Western Blot Analysis of Cell Cycle Regulators
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The total protein of each group was harvested and collected using RIPA lysis buffer (Thermo Fisher Scientific, Inc.). Protein concentration was measured with a BCA Protein Assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein (30 µg/lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (EMD Millipore). The membrane was blocked by incubation with 5% non-fat milk for 1 h at 24°C and then with primary antibodies overnight at 4°C. The primary antibodies used were: Anti-DACH1 (cat. no. ab176718; Abcam; 1:1,000), anti-Cyclin D1 (cat. no. 55506; Cell Signaling Technology, Inc.; 1:1,000), anti-Cyclin A (cat. no. 91500; Cell Signaling Technology, Inc.; 1:1,000) and anti-P21 (cat. no. 2947; Cell Signaling Technology, Inc.; 1:1,000), anti-P53 (cat. no. 2527; Cell Signaling Technology, Inc.; 1:1,000). After washing three times with Tris buffered saline-Tween (TBS-T; 50 mM Tris, 150 mM NaCl, 0.05% Tween-20) the membranes were incubated with goat anti-rabbit (cat. no. ab6721; Abcam; 1:5,000) or anti-mouse (cat. no. ab205719; Abcam; 1:5,000) IgG/horseradish peroxidase secondary antibody at room temperature for 1 h. Protein expression was detected using a chemiluminescence detection system (Tanon 4600SF; Tanon Science and Technology Co., Ltd.).
8
Liver Protein Expression Analysis
9
Biochanin A Modulates Cell Cycle Proteins
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A549 and 95D cells were treated with various concentrations of Biochanin A (0, 50, 100, and 200 μmol/L for A549 and 0, 60, 120, and 240 μmol/L for 95D) for 48 h; then A549 and 95D cells and the previous tumor tissues were lysed in lysis buffer (RIPA, 1 mM PMSF) and phosphatase inhibitors (Roche, Germany) to extract the total protein. Proteins were separated by SDS-PAGE (8%, 12% gels) under reducing conditions. The proteins were then electrophoretically transferred to nitrocellulose membranes. The membranes were blocked with 5% skimmed milk and incubated with anti-cyclin A, anti-P21, anti-Caspase-3, anti-Bcl-2, anti-Bax, anti-CDK2, and anti-β-actin antibodies, respectively (1:1000; Cell Signaling Technology) overnight at 4°C. This was followed by an incubation with goat anti-rabbit/anti-mouse secondary antibody (1:5000; Abcam). An equal loading of each lane was evaluated by immunoblotting the same membranes with anti-β-actin antibodies after the detachment of previous primary antibodies. The chemiluminescence reaction was detected by an ECL detection kit and the results were analyzed with the Gel Doc 2000 (Bio-Rad, USA).
10
Cordycepin-Induced Apoptosis Pathway
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Cordycepin was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, People’s Republic of China). The primary and secondary antibodies used for Western blotting, such as rabbit anti-Bcl-2, anti-Bax, anti-cleaved-caspase-3, anti-cleaved-caspase-9, anti-cleaved PARP, anti-Cdk-2, anti-Cyclin A, and mouse anti-β-actin, were purchased from Cell Signaling Technology (Beverly, MA, USA).
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