Whole cell lysates were prepared in a buffer comprised of (in mM) 100 Tris-HCl, 2 EDTA, 2 EGTA, 1 PMSF, 1 benzamidine, 1 dithiothreitol, 1 Na3VO4, 20 NaF, 10 Na2MoO4, 10 Na-pyrophosphate and 50 β-glycerol phosphate and supplemented with a GBioscience PhosphataseArrest™ III inhibitor cocktail, Roche complete proteinase inhibitor tablet, 10 μg/ml pepstatin A and 4% (w/v) SDS (pH 7.2). Lysates were resolved by SDS-PAGE on a 10 or 12% polyacrylamide gel and transferred to PVDF or nitrocellulose membranes. Membranes were blocked for 1 h in Tris-buffered saline ( pH 7.5) containing 3% (w/v) BSA and 0.1% (v/v) Tween-20. The membranes were incubated with primary antibodies overnight at 4°C. Rabbit polyclonal antibody against MCU/CCDC109A was obtained from Sigma, rabbit polyclonal antibody against voltage-dependent anion channel (VDAC) was from Cell Signaling, mouse monoclonal antibody against NDUFA9 was from Molecular Probes, mouse monoclonal antibodies against CypD/CypF and mitochondrial respiratory chain polypeptides were from Abcam and rabbit monoclonal antibodies against Hsp10 and Hsp60 were from Epitomics. Protein loading was determined by stripping the membranes and reprobing with anti-α-tubulin or anti-β-actin antibodies (Cell Signaling). The levels of α-tubulin or β-actin did not change with alcohol feeding and either protein was used as a loading control.
Mouse monoclonal antibodies
Manufactured by Abcam
Sourced in United Kingdom, United States
Mouse monoclonal antibodies are laboratory-produced antibodies derived from mouse B cells. They are highly specific to a targeted antigen and can be used in various research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Maxisorp microtitre plate, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Merck Group (2 mentions)
Anti β actin antibody, by Cell Signaling Technology (1 mentions)
Anti α tubulin, by Cell Signaling Technology (1 mentions)
Mannitol, by Merck Group (1 mentions)
Collagenase nb4, by Serva Electrophoresis (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Kaiser s glycerol gelatin, by Merck Group (1 mentions)
Mayer s hematoxylin solution, by Merck Group (1 mentions)
Cell culture media, by Euroclone (1 mentions)
Acetone, by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ab8925, by Abcam (1 mentions)
Envision system, by Agilent Technologies (1 mentions)
Mouse monoclonal antibody against β actin, by Santa Cruz Biotechnology (1 mentions)
Coomassie blue, by Merck Group (1 mentions)
Pd98059, by Cell Signaling Technology (1 mentions)
Endoglycosidase h endo h, by Roche (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
16 protocols using mouse monoclonal antibodies
1
Protein Expression Analysis in Alcoholic Cells
2
Porous Scaffold Characterization and Cell Culture
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Cell culture media and antibiotics were purchased from Euroclone (Milan, Italy). A commercial platelet lysate kit (PL) was obtained from Carlo Erba Reagents (Milan, Italy). Collagenase NB4 was purchased from SERVA Electrophoresis, Heidelberg, Germany. MicroDec EDTA-based was purchased from Diapath, Martinengo, Italy. The EZ Prep Concentrate solution and the Universal DAB Detection Kit were purchased from Ventana Medical Systems Inc., Tucson, AZ, USA. Mouse monoclonal antibodies were obtained from Abcam (Milan, Italy). Acetone, beta-glycerophosphate, bovine serum albumin (BSA), formaldehyde, Kaiser’s glycerol gelatin, Mayer’s hematoxylin solution, mannitol, phosphate-buffered saline (PBS), phosphatidylcholine (PC), poloxamer 407 (Lutrol® F127), and sodium chloride were purchased by Sigma Aldrich (Milan, Italy). All reagents were of analytical grade. SmartBone® scaffolds (1 cm × 1 cm × 0.3 cm in size) were provided by Industrie Biomediche Insubri SA (Mezzovico-Vira, Switzerland).
3
Immunohistochemical Analysis of Colon Cancer
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Colon cancer sections were obtained from Nagoya City University Hospital (Nagoya, Japan). For antigen retrieval, deparaffinized sections were boiled in citrate buffer (10 mM sodium citrate buffer, pH 6.0) prior to incubation with the primary antibodies. NOTCH1 and β-catenin protein levels were examined using rabbit polyclonal antibodies (ab8925) and mouse monoclonal antibodies (Abcam, Cambridge, UK). Nuclear CyclinD1 and HistonH3 protein levels were examined using rabbit monoclonal antibodies (Cell Signaling Technology, Inc. Dancers, MA, USA)
Antibody staining was conducted using the peroxidase-based DAKO EnVision System.
Antibody staining was conducted using the peroxidase-based DAKO EnVision System.
4
EMMPRIN Regulation via ERK/IκB Pathway
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Fetal bovine serum (FBS) and Medium 199 (M-199) were purchased from Gibco BRL (Life Technologies, NY). Phorbol 12-myristate 13-acetate (PMA), H2O2, peptide-N-glycosidase F (PNGase F), swainsonine, calcein-AM and Coomassie blue were acquired from Sigma-Aldrich (St. Louis, MO); Endoglycosidase H (Endo H) from Hoffmann-La Roche (Basel Switzerland); TNF-α from eBioscience (San Diego, CA); PD98059 from Cell Signaling (Danvers, MA). For protein purification, the Immunopure rprotein A IgG plus orientation Kit and the silver staining kit were provided by Pierce (Rockford, IL); and the monoclonal antibody against EMMPRIN (mAb-EMMPRIN) was produced by Genscript Corporation (Nanjing, China). For Western blot, the protein extraction kit was acquired from Beyotime (Haimen, China); mouse monoclonal antibodies against EMMPRIN, pERK1/2, pIκBα, ERK1/2, IκBα and rabbit polyclonal antibodies against MMP9 from Abcam (Cambridge, MA); the mouse monoclonal antibody against β-actin from Santa Cruz (Dallas, Tex).
5
Immunohistochemical Analysis of Glioma Samples
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Formalin-fixed paraffin-embedded glioma blocks were serially sectioned at a 4 µm thickness and deparaffinized in xylene. The sections were rehydrated after immersing in ethanol and incubated with goat serum to block nonspecific binding. The sections were then incubated overnight at 4 °C with primary rabbit monoclonal antibodies against NLRP3 and CARD12 or with mouse monoclonal antibodies against ionized calcium-binding adaptor protein 1 (Iba1) or glial fibrillary acidic protein (GFAP) (Abcam, Cambridge, MA). Secondary antibodies were applied for 1 h at room temperature. Images were acquired using a confocal laser scanning microscope (Zeiss LSM 880; Carl Zeiss, Jena, Germany).
6
Enzyme-Linked Immunosorbent Assay for Plasma Proteins
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Plasma samples (50ul) with different dilutions as recommended by the manufacturers were added to 96-well EIA plates coated with mouse monoclonal antibodies (Abcam Inc., Cambridge, UK). The wells were incubated at 25 °C for 30 min. After washing twice, the secondary antibody of goat anti-mouse immunoglobulin antibody conjugated with horseradish peroxidase (HRP) (Santa Cruz Biotech. Inc., Santa Cruz, CA) was added to the reaction for 30 min. The reactions were visualized by OD550nm (Pierce, Rockford, IL) as previously described [15 (link)]. Concentrations of plasma proteins were determined by the interpolation of a standard curve made from a series of well-known concentrations of standard samples [15 (link)].
7
Fibroblast Isolation and Characterization
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Fascia was digested with Collagenase B 0.1% in Hank’s Balanced Salt Solution (HBSS) overnight and fibroblast cells were isolated and characterized by immunohistochemical staining with anti-Fibroblast Surface Protein [1B10] antibody (1:100, Mouse monoclonal antibodies; Abcam, Cambridge, UK), as previously described in one of our previous works.12 (link) Cell cultures were maintained at 37°C, 95% humidity and 5% CO2, in DMEM 1g/L glucose, 10% FBS and 1% penicillin-streptomycin antibiotic, and used from passage 3rd to 9th.
8
Protein Expression Analysis in Virus-Infected Cells
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Virus-infected Vero cells or their culture supernatants were lysed in 1× RIPA buffer (G-Bioscience) containing 1% PMSF (Theromfisher Scientific) at 4 °C. Fifty μg of protein from each cell lysate was resolved on denaturing polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Amersham). Expression levels of PA and CTA1 proteins were detected using mouse monoclonal antibodies against PA and cholera toxin, respectively (Abcam, 1:1000), and horse radish peroxide (HRP)-conjugated secondary antibodies (Abcam, 1:3000).
9
Quantifying Alpha-1 Proteins in ELS
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Alpha-1-fetoprotein and alpha-1-antitrypsin production were quantified by sandwich ELISA in ELS conditioned media collected 1–3 days post-thaw. This was normalized with cell counts and compared to an unfrozen control.
Mouse monoclonal antibodies (Abcam, Cambridge, UK cat # ab10071 and ab10072) were used as a capture and as an HRP linked antibody respectively, with Applichem (cat # A6935) used for a standard curve.
Mouse monoclonal antibodies (Abcam, Cambridge, UK cat # ab10071 and ab10072) were used as a capture and as an HRP linked antibody respectively, with Applichem (cat # A6935) used for a standard curve.
10
Placental JNK Expression Analysis
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The sections of placenta were deparaffinization and rehydration by xylene, 100%/95% ethanol and distilled water. Then, all procedures were performed as Immunohistochemistry Protocol (Paraffin) from the Cell Signaling Technology and incubated overnight at 4 °C with mouse monoclonal antibodies against JNK (Abcam, state abbreviation, USA), then added to biotinylated rabbit anti-mouse IgG secondary antibody. Diaminobenzidine (DAB) as a chromogenic agent showed a positive result. Finally, all slides were imaged under microscopy. Image J 6.0 was used to evaluate the images.
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