Whole protein extracts were obtained using T-PER Tissue Protein Extraction Reagent (ThermoFisher) supplemented with Complete O, Mini, EDTA-free Protease Inhibitor Cocktail (Roche Applied Science), and the concentration of total proteins was determined using BCA Protein Assay Reagent (Pierce). A quantity of 60 µg of total proteins for each sample was electrophoretically separated by SDS-PAGE and transferred onto PVDF membranes that were subsequently blocked in Tris-buffer saline - 0.5% Tween 20 with 2% bovine serum albumin and then incubated with the primary antibodies against COL10A1 and β-actin at 4 °C overnight. The antibodies used were rabbit polyclonal anti-collagen X (Abcam, ab58632, 1:500 dilutions) and mouse monoclonal anti-b-actin clone Ac-74 (Sigma Aldrich, 1:1000 dilution). Proteins of interest were detected with the appropriate secondary antibodies (1:1000 dilutions) conjugated with HRP: anti-rabbit IgG (RD Systems), and anti-mouse IgG (RD Systems). Signals were developed using ECL HRP chemiluminescent substrate (Invitrogen) and captured using a MicroChemi 4.2 system (Bio-Imaging Systems).
Mouse monoclonal anti b actin clone ac 74
Manufactured by Merck Group
Sourced in United Kingdom
Mouse monoclonal anti-b-actin clone Ac-74 is a laboratory reagent used for the detection and quantification of the beta-actin protein in various biological samples. It is a mouse-derived monoclonal antibody that specifically binds to the beta-actin isoform.
Automatically generated - may contain errors
Lab products found in correlation
Ab58632, by Abcam (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Ecl hrp chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Fluorochrome conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Pegfp c1, by Takara Bio (1 mentions)
Ab56307, by Abcam (1 mentions)
Ab51074, by Abcam (1 mentions)
2 protocols using mouse monoclonal anti b actin clone ac 74
1
Whole Protein Extraction and Western Blotting
2
Plasmids and Antibodies for MMP14 Study
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The pEGFPc1 (Clontech, Mountan View, CA, USA) plasmids encoding for the wild-type form of PTP4A3 (pEGFP-PTP4A3) or the catalytic mutant (pEGFP-PTP4A3(C104S)) were both kindly provided by Qi Zeng (IMCB Institute, Singapore) and were first described by Zeng et al. 25 The MMP14-mCherry construct was kindly provided by P. Chavrier (Institut Curie, Paris, France). Rabbit monoclonal or mouse monoclonal anti-MMP14 antibodies (ab51074 and ab56307, respectively) were purchased from Abcam (Cambridge, UK), and mouse monoclonal anti-b-actin (clone AC-74) was purchased from Sigma-Aldrich. All fluorochrome-conjugated secondary antibodies were purchased from Molecular Probes (Eugene, OR, USA).
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