Total cellular proteins were extracted, quantified, and subjected to gel electrophoresis according to standard procedures as we described previously [35 (link)]. The antibodies used in this study included anti-HO-1 (ab52947; Abcam, Cambridge, UK), anti-Cyclin D1 (#2978), anti-CDK4 (#12790), anti-p21 Waf1/Cip1 (#2947), anti-p27 Kip1 (#3686), and anti-actin (A5441; Sigma-Aldrich). Antibodies were obtained from Cell Signaling Technology, Danvers, MA, USA, unless specified otherwise. Immunoreactive bands were detected by enhanced chemiluminescence (Amersham ECL Detection System; GE Healthcare, Piscataway, NJ, USA).
Anti cyclin d1
Manufactured by Merck Group
Sourced in United States, Germany
Anti-Cyclin D1 is a laboratory reagent used in research applications. It is an antibody that specifically binds to the Cyclin D1 protein, which is involved in the regulation of the cell cycle. This product can be used in various experimental techniques, such as Western blotting, immunohistochemistry, and flow cytometry, to detect and analyze the expression of Cyclin D1 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti cdk4, by Merck Group (3 mentions)
Anti actin, by Merck Group (3 mentions)
Anti c myc, by Merck Group (3 mentions)
Anti p21, by Merck Group (3 mentions)
Anti p27 kip1, by Merck Group (2 mentions)
Anti β catenin, by Merck Group (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti ha, by Merck Group (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti α tubulin, by Merck Group (2 mentions)
Anti p84, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti gapdh, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ab52947, by Abcam (1 mentions)
Anti p21 waf1 cip1, by Merck Group (1 mentions)
Amersham ecl detection system, by GE Healthcare (1 mentions)
Anti e cadherin, by Merck Group (1 mentions)
21 protocols using anti cyclin d1
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Cell Signaling Proteins
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The cells were pyrolysis in RIPA buffer containing protease inhibitors. The lysates were centrifuged at 10,000 rpm for 10 min at 4°C. After centrifuge, the supernatant were collected. Total of 50 µg protein was uploaded to sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto a PVDF membrane. The membrane was blocked using 5% fat-free milk at room temperature for 2 h. The concentration of antibody for incubated is as following: anti-E-cadherin (1:200), anti-Cyclin D1 (1:1,000), anti-C-Myc (1:1,000), anti-β-catenin (1:500), anti-β-actin (1:1,000) (Sigma, USA) at 4°C for overnight. After three washes for 5-min in TBST, the membrane was incubated in peroxidase-conjugated goat anti-mouse/rabbit IgG or peroxidase-conjugated rabbit anti-goat IgG (Abcam, US) for 2 h at room temperature. An electrogenerated chemiluminescence system was used to visualize the proteins.
3
Western Blot Assay for Protein Detection
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For Western blotting, cell lysates in ice-cold RIPA buffer were centrifuged and the supernatants were assayed for protein content. About 20 to 30 μg of proteins were fractionated on 7.5–15% polyacrylamide gel and transferred onto PVDF membrane from Millipore Corporation (Billerica, MA, USA). Nonspecific binding sites were blocked for 1 hour at RT in 20 mM Tris-HCl (pH 7.4) buffer, 55 mM NaCl and 0.1% Tween 20 containing 5% non-fat dry milk (blocking buffer). Membranes were then incubated overnight at 4°C with primary antibody diluted in blocking buffer. Primary antibodies used are anti-LC3, anti-cyclin D1, anti-p62, anti-actin and anti-pERK1,2 antibodies from Sigma Aldrich (St. Louis, USA), anti-p53 and anti-pEGFR antibodies from Santacruz Biotectnology (SantaCruz, CA, USA), anti-cyclin B2 was from Abcam (Cambridge Science Park, Cambridge, UK). All these antibodies are dissolved in blocking solution. After extensive washings and incubation with the respective horseradish peroxidase-labeled secondary antibodies, protein presence was visualized by enhanced chemiluminescence reaction from Pierce Biotechnology (Rockford, IL, USA). Band relative densities obtained using Alliance 4.7 UVITEC (Cambridge, UK) were normalized to actin and values were given as relative units (R.U.).
4
Immunofluorescence Staining of Neural Markers
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Control and treated cells were given a washing with chilled 1X PBS followed by fixation with acetone and methanol (1:1) and permeabilization with 0.3% Triton- X100 in 1X PBS. Cells were then blocked with 2% BSA and incubated with primary mouse monoclonal antibody anti-α-Tubulin (1:500), anti-NF-κB (1:500), anti-MAP-2 (1:250), anti-NF200 (1:500), anti-GAP 43 (1:250), anti-HSP70 (1:500), anti-Mortalin (1:500), anti-Bcl-xL (1:200), anti-Cyclin D1(1:250), anti-NCAM (1:250), rabbit monoclonal anti-AP-1(1:250) (all from Sigma-Aldrich) and mouse monoclonal anti-PCNA (1:250), mouse polyclonal anti-PSA-NCAM (1:250) (from Millipore, MA, USA) for 24 h in humid chamber at 4 °C. No permeabilization was carried out for PSA-NCAM immunostaining. After primary antibody incubation, three washings were given with 0.1% PBST and incubated with secondary antibody (goat anti-mouse/ rabbit IgG/ IgM Alexa Fluor 488/543) for 2 h at RT. Cells were stained with nuclear staining dye DAPI (Sigma-Aldrich) for 15 min, washed with 0.1% PBST and mounted with antifading agent Fluoromount (Sigma-Aldrich). Images were captured with Nikon AIR Confocal Laser Scanning Microscope and analyzed with NIS elements analysis software version 4.11.00 (Nikon Co., Tokyo, Japan). Each experiment was carried out in triplicate.
5
Western Blot Analysis of Cell Signaling
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Western blots were done as previously described [37 (link)], with the primary antibodies: anti-AKT (11E7), anti-phospho-AKT(Ser473), anti-p65 NFκB subunit (E498), anti-phospho-p65 (Ser536), anti-IκBα (L35A5), anti-caspase 3,anti-caspase 9, anti-c-Myc and anti-phospho-GSK3αβ (Ser21,9) from Cell Signaling Technology; anti-BCL2, anti-BAX (6a7), anti-survivin, anti-cyclin D1 and anti-β-actin (AC-15) from Sigma; anti-BCL-XL and anti-PARP (C2-10) and anti-p53 from BD Biosciences; anti-phospho-histone H2A.X (Ser139) and anti-cIAP (315301) from R&D Systems and anti-GSK3β from Santa Cruz Biotechnologies.
6
Western Blotting for Protein Analysis
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Western blotting was performed according to standard methods as previously described (14 (link)). Cells and tissues were lysed with radioimmunoprecipitation assay lysis buffer (Biyuntian Biotechnology Research Institute, Nantong, China). The protein concentration of each lysate was determined with the BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Total proteins (30 µg/lane) were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. The blots were blocked in 5% milk for 1 h at room temperature. PVDF membranes were incubated using rabbit anti-LASP2 (1:100; cat. no. 260630; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), anti-cyclin D1 (1:10,000; cat. no. ab134175; Abcam, Cambridge, UK) and anti-β-catenin (1:1,000; cat. no. ab22656; Abcam) antibodies overnight at 4°C. Subsequently, membranes were incubated with corresponding horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:5,000; cat. no. ab6112; Abcam) for 2 h at room temperature and detected using an enhanced chemiluminescence reagent (ECL Prime; GE Healthcare, Chicago, IL, USA). The anti-α-tubulin (1:5,000; cat. no. ab4074; Abcam,) and anti-P84 (1:10,000; cat. no. ab131268; Abcam) antibodies were used as loading controls.
7
Quantitative Western Blot Analysis
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The cell protein lysates were separated via SDS-PAGE, and were transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Then they were incubated at 4°C overnight in the presence of anti-HMGA1 (Cell Signaling Technology, Boston, MA, USA), anti-P21 (Sigma-Aldrich Co., St Louis, MO, USA), and anti-cyclin D1 (Sigma-Aldrich Co.), respectively. After washing, secondary antibody (Pierce, Rockford, IL, USA) was added into the system. Enhanced chemiluminescence chromogenic substrates were used for quantitative measurement by using Quantity One software (Bio-Rad). GAPDH was used as an internal control.
8
Western Blot Protein Analysis
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Proteins were extracted using RIPA buffer (Sigma), supplemented with protease (1:1000, Sigma) inhibitor cocktails, after 30 min incubation on ice. Protein concentrations were measured using BCA assay, with BSA for the standard curve, and 50 μg of protein were resolved on 12% NuPAGE gels (Invitrogen) and transferred using a semi-dry transfer system onto PVDF membranes (Millipore). Non-specific binding sites were blocked with 10% milk TBS-T solution for 1 h at room temperature, then probed overnight at 4 °C with the respective primary antibodies: anti-SAMHD1 (Abcam), anti-SOX11 (Sigma), or anti-Cyclin D1 1:1000 in 5% milk or 5% BSA in TBS-T. Membranes were then washed in TBS-T and probed with secondary antibodies (HRP-conjugated anti-rabbit or anti-mouse; GE Healthcare). Blots were developed using Supersignal West Pico (Pierce) and visualized using LiCor machine. For re-probing of membranes with anti-actin (Sigma) or anti-GAPDH (Cell Signaling) (1:5000 in 5% milk in TBS-T, Sigma), HRP was blocked using the SG substrate kit (Vector Labs). Analysis was done using Fiji-ImageJ software.
9
Western Blot Analysis of Cellular Proteins
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Cells were lysed in 50 μl radio immunoprecipitation assay (RIPA) buffer. Equal amounts of total cell lysates were boiled in SDS-sample buffer and later separated by SDS-PAGE at 100 V for 3 h. Proteins on the gel were transferred to a PVDF membrane at 100 V for 1.5 h at 4°C and the membrane was blocked with 5% milk in 1 x TBST (Tris-buffered saline, 0.1% Tween 20) buffer at room temperature for 1 h. Blots were subsequently incubated with a primary antibody and a peroxidase-conjugated secondary antibody. After washing five times with TBST, blots were incubated with enhanced chemiluminescence (ECL) detection reagent, and imaged with the ChemiDoc MP (Bio-Rad, USA). The primary antibodies used for the blots were anti-GAPDH (Sigma, USA), anti-Flag (Sigma, USA), anti-HA (Sigma, USA), anti-β-catenin (Sigma, USA), anti-FOXM1 (Abcam, USA), anti-c-Myc (Sigma, USA) and anti-Cyclin D1 (Sigma, USA).
10
Western Blot Analysis of Cell Signaling Proteins
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Following treatments, cells were washed twice with PBS and lysed in 1× SDS sample buffer. Proteins were separated on 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were washed twice with tween Tris-buffered saline before blocking nonspecific binding with 5% nonfat dry milk (Blotto, Santa Cruz Biotechnology, Dallas, TX, USA) or 3% bovine serum albumin. Anti-Poly ADP-ribose Polymerase (PARP) (11835238001-Roche) antibody, that detects total and cleaved PARP was used at 1:1000 dilutions, and membranes were incubated for 2 h at room temperature. Anti-Cyclin D1 (C7464, Sigma Aldrich) and anti-CDK4 (SAB140559, Sigma Aldrich) were detected similar to PARP protein. Membranes were washed three times, and detection was performed using horseradish peroxidase-conjugated secondary antibody as described previously [40 (link)].
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