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7 protocols using mouse anti cyclin a

1

Western Blot Analysis of Cell Signaling

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Protein concentration was determined with the Bio-Rad DC protein assay. Protein samples were resolved on 10% or 13% polyacrylamide gels in an SDS running buffer. After incubation with the primary antibody and the corresponding horseradish peroxidase (HRP)–coupled secondary antibody, protein signals were detected by enhanced chemiluminescence (ECL) with a digital image analyzer ImageQuant LAS4000 from GE Healthcare, Chicago, IL, USA or IMAGEQUANT 800. The following antibodies were used: rabbit anti–p27Kip1–C19 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti–p27Kip1–horseradish peroxidase–coupled antibodies (clone 57, BD Biosciences, Franklin Lakes, NJ, USA), mouse anti–pY88–p27Kip1 [14 (link)], rat anti–EpoR (GM1201, clone: BCO–3H2), rat anti–EpoR (GM1204) [22 (link)], mouse anti–Jak2 (clone 691R5, Invitrogen, Carlsbad, CA, USA), rabbit anti–pY1007/1008–Jak2 (Millipore, Burlington, MA, USA), rabbit anti–pY396 Lyn (Labome/Epitomics, Burlingame, CA, USA), rabbit anti–pY694 STAT5 (Cell Signaling, Danvers, MA, USA), mouse anti–Cyclin A (clone E67.1, Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti–Cyclin E (clone HE12, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti–Cyclin A polyclonal antibody T310 [23 (link)], mouse anti–GAPDH (clone 6C5, Millipore, Burlington, MA, USA), and mouse monoclonal anti–PSTAIR [24 (link)].
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2

Immunofluorescence Staining of HeLa and Flp-In T-REx 293 Cells

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HeLa Kyoto cells or Flp-In T-REx 293 cells were fixed and processed for immunofluorescence as described45 (link). In brief, cells were fixed in 4% paraformaldehyde for 10 min at the room temperature followed by ice-cold methanol for 1 min. The cells were then wash with 1 × phosphate-buffered saline (PBS) and permeabilized with 0.5% Triton X-100 for 5 min before antibody staining. The primary antibodies used were as follows: mouse anti-histone H2AX pSer139 (1:1,000, Millipore 05-636-1), mouse anti-α-tubulin (1:5,000, Sigma-Aldrich T9026), rabbit anti-GFP (1:5,000, Abcam ab290), mouse anti-cyclin A (1:200, Santa Cruz sc-56299), mouse anti-FLAG (1:1,000, Sigma-Aldrich A8592). Secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 555 (1:1,000, Molecular Probes) were used for detection. DNA was stained with 4,6-diamidino-2-phenylindole. Images were acquired using Zeiss AXIO Imager M1 with a × 40 EC-Plan-Neofluor lens and Hamamatsu photonics camera under the control of Volocity software. Images were processed using Adobe Photoshop.
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3

Immunofluorescence Imaging of Cell Markers

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HeLa Kyoto cells or Flp-In T-REx 293 cells were fixed and processed for immunofluorescence as described45 (link). In brief, cells were fixed in 4% paraformaldehyde for 10 min at the room temperature followed by ice-cold methanol for 1 min. The cells were then wash with 1xPBS and permeabilised with 0.5% Triton X-100 for 5 min before antibody staining. The primary antibodies used were: mouse anti-histone H2AX pSer139 (1:1000, Millipore 05-636-1), mouse anti-α-tubulin (1:5000, Sigma-Aldrich T9026), rabbit anti-GFP (1:5000, Abcam ab290), mouse anti-cyclin A (1:200, Santa Cruz sc-56299), mouse anti-FLAG (1:1000, Sigma-Aldrich A8592). Secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 555 (1:1000, Molecular Probes) were used for detection. DNA was stained with DAPI. Images were acquired using Zeiss AXIO Imager M1 with a 40x EC-Plan-Neofluor lens and Hamamatsu photonics camera under the control of Volocity software. Images were processed using Adobe Photoshop.
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4

Immunoblotting Analysis of Cellular Proteins

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The proteins were separated with gel electrophoresis, using Novex Bis-Tris 4–12% gels, MES buffer, NuPAGE LDS loading buffer and NuPAGE reducing agent according to the manufacturer's recommendations (ThermoFisher). The proteins were then transferred to a PVDF membrane using a Trans-Blot Turbo system (1.3 A, 25 V for 7 min) and the RTA lf-PVDF kit (Bio-Rad) according to the manufacturer's instructions. Antibody incubations were performed with Super Signal West Femto Rabbit or Mouse kit (ThermoFisher) along with primary antibody incubations overnight: rabbit anti-Atox1 (1 μg/mL, Abcam), mouse anti-GADPH (2 μg/mL, Abcam), mouse anti-cyclin B1 (1 μg/mL, Santa Cruz) or mouse anti-cyclin A (1 μg/mL, Santa Cruz), and detected with ChemiDoc MP (Bio-Rad) using high sensitivity chemiluminescence detection.
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5

Immunoblot Analysis of Cell Signaling

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Preparation of lysates and immunoblot analyses were performed as described previously45 (link) using Tris lysis buffer (50 mM Tris–HCl pH 7.8, 150 mM NaCl, 1% IGEPAL CA-630) containing 100 mM NaF, 100 mM β-glycerophosphate, 20 μg/ml RNase A, 20 μg/ml DNase and 1/300 protease inhibitor cocktail (P8340, Sigma–Aldrich) and 1/100 phosphatase inhibitor cocktail #2 (P5726, Sigma–Aldrich). Antibodies used in this study were purchased from the following sources: rabbit anti-Cdk2 (M2 SC-163, Santa Cruz Biotechnology), mouse anti-tubulin (Developmental Studies Hybridoma Bank, University of Iowa), mouse anti-Cyclin A (SC-751, Santa Cruz Biotechnology), mouse anti-cyclin E (SC-198, Santa Cruz Biotechnology), rabbit anti-CDK4 (sc-260, Santa Cruz Biotechnology), mouse anti-Rb (G3-245, BD Pharmingen), rabbit anti-p27 (sc-528, Santa Cruz Biotechnology) and anti-HA High affinity (3F10, 11867423001, Sigma-Aldrich). Secondary antibodies used for western blot analysis were goat anti-mouse (31430, Thermo Scientific) and, goat anti-rabbit (31460, Thermo Scientific). mouse anti-tubulin hybridoma cell line (clone #12G10) was developed by J. Frankel and E.M. Nelson under the auspices of the NICHD and maintained by the Developmental Studies Hybridoma Bank.
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6

Western Blot Quantification of Apoptosis-Related Proteins

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From protein extraction, quantitation, separation by 12 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transfer to polyvinylidene fluoride membrane, and to protein visualization, Western blot technology was employed as previously described (Matou-Nasri et al., 2022 (link)). Diluted in blocking buffer, the primary antibodies used were rabbit anti-cleaved caspase-3 (#9664, dilution 1:1000), pro-caspase 3 (#9665, 1:1000), cleaved caspase-9 (#9505, 1:1000), cleaved poly (ADP-ribose) polymerase (PARP) (#5625, 1:500), PARP (#9542, 1:500), mouse pro-caspase-9 (#9508, 1:1000) monoclonal antibodies provided by Cell Signaling Technology (Danvers, MA, USA), and mouse anti-cyclin A (#sc-271645, 1:1000), cyclin B1 (#sc-70898, 1:1000), cyclin D1 (#sc-8396, 1:1000) monoclonal antibodies purchased from Santa Cruz Biotechnology, and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (#AM4300, 1:5000) from Invitrogen.
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7

Antibody Generation and Characterization

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Rabbit anti-human SUN1 (Atlas) and mouse monoclonal β-actin antibodies were obtained from Sigma. Mouse anti-Myc antibody was purchased from Zymed Laboratories Inc Mouse anti-cyclin B1, mouse anti-cyclin A and goat anti-lamin A/C (6215) antibodies were purchased from Santa Cruz Biotechnologies. Rabbit anti-GFP antibody was obtained from Abcam. Rabbit anti-emerin antibody was kindly provided by G. Morris (Center for Inherited Neuromuscular Disease, Oswestry, UK). Anti-human SUN1 2383 has been reported previously.27 (link)
SUN1 serine 48 (pS48) phospho-antibodies were generated by immunization of rabbits with a peptide corresponding to residues 42–55 of human SUN1, phosphorylated at serine 48. Immunizations and antibody purification, using peptide columns, were performed by Eurogentec.
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