Protein concentration was measured using a BCA Protein Assay Kit following the manufacturer’s protocol (Thermo Fisher Scientific). Proteins (25 μg/sample) were separated on a 4–12% SDS-PAGE gel and transferred onto nitrocellulose membrane blots using semi-dry blotting system (Bio-Rad) following the manufacturer’s protocol. To ensure the equal protein loading in each lane, the blots were stained Ponceau S (Sigma) and imaged on a ChemiDoc™ imaging system (Bio-Rad). Blots were then incubated with primary monoclonal/polyclonal antibodies including CFD (R&D systems, AF1824, 1:2500), GPX3 (R&D systems AF4199, 1:200), CST3 (Abcam ab133495, 1:13000), PON1 (Abcam, ab92466, 1:5000), MRC1 (Abcam ab195193, 1:1000) and COMP (Abcam, ab74524, 1 :200), followed by respective HRP-conjugated secondary antibodies. Blots were imaged using a Li-Cor Odyssey Blot imager (LI-COR Biosciences). Quantitation of signal intensity of the bands in Western blots was performed using Image lab software version 5.0 (Bio-Rad) and Image Studio Lite version 5.2 (LI-COR Biosciences).
Ab74524
Manufactured by Abcam
Ab74524 is a lab equipment product. It is a core component used for sample preparation and analysis in various scientific experiments and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab11306, by Abcam (2 mentions)
Ab96569, by Abcam (2 mentions)
Ab75769, by Abcam (2 mentions)
Ab76956, by Abcam (2 mentions)
Ab108337, by Abcam (2 mentions)
Ab34710, by Merck Group (2 mentions)
Ab33734, by Abcam (2 mentions)
Ab189267, by Abcam (2 mentions)
Ab203887, by Abcam (2 mentions)
Ab185716, by Abcam (2 mentions)
Ab107595, by Abcam (2 mentions)
Ab93876, by Abcam (2 mentions)
Ab19027, by Abcam (2 mentions)
Mab8887, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab133495, by Abcam (1 mentions)
Ab92466, by Abcam (1 mentions)
Image studio lite version 5, by LI COR (1 mentions)
Af1824, by R&D Systems (1 mentions)
Odyssey blot imager, by LI COR (1 mentions)
5 protocols using ab74524
1
Quantitative Western Blot Analysis
2
COMP Protein Immunofluorescence and STORM Analysis
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The commercial antibodies used in this study, and their sources, are as follows: Anti-COMP (rabbit polyclonal, ab74524; Abcam), anti-FLAG (mouse monoclonal, F3165; Sigma-Aldrich), and anti-TGN46 (sheep polyclonal, AHP500G; AbD Serotec) were used for WB, together with the secondary antibodies sheep (sc-2770, sc-2303, rabbit sc-2301 and mouse sc-2314) obtained from Santa Cruz Biotechnology, Inc. Anti-HA (rat monoclonal, 11867423001; Roche), anti-FLAG (mouse monoclonal; F3165; Sigma-Aldrich), anti-GM130 (mouse monoclonal, 610822; BD Biosciences), and anti-ATP2C1 (mouse monoclonal, HPA035116; Sigma-Aldrich) were used for immunofluorescence microscopy. The secondary antibodies Alexa Fluor 488 rabbit (A21206), Alexa Fluor 594 mouse (A21203), and Alexa Fluor 488 rat (A21208) were purchased from Thermo Fisher Scientific. For STORM measurements Cy5-conjugated AffiniPure (rabbit, 711–175-152; Jackson ImmunoResearch Laboratories, Inc.) was used for Cab45 as a secondary antibody and Alexa Fluor 488 sheep (A11015) for TGN46 (Thermo Fisher Scientific). The recombinant proteins COMP-FLAG (COMP-3659H; Creative BioMart), COMP (ab104358; Abcam), LysozymeC (Sigma-Aldrich), and LysozymeC-Cy5 (LS1-S5-1; Nanocs) were used for in vitro analysis.
3
Characterization of Musculoskeletal Cell Markers
4
Characterization of Musculoskeletal Cell Markers
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Primary antibodies used were specific for Collagen type I (Abcam, ab34710, 1:100 dilution), Collagen type II (Millipore, MAB8887, 1:100 dilution), murine CD200 (Abcam, ab33734, 1:100 dilution), Nestin (Abcam, ab11306, 1:200 dilution), Gremlin 1 (Abcam, ab189267, 1:50 dilution), COMP (Abcam, ab74524, 1:50 dilution), Aggrecan (Abcam, ab3778, 1:100 dilution), Thy1.2 (Invitrogen, cat no. 14-0902-82, 1:50 dilution, 6C3 (Invitrogen, cat no. 14-5891-82 (1:50 dilution), CD105 (Abcam, ab107595, 1:100 dilution), Runx2 (Abcam, ab76956, 1:200 dilution), Alpl (Abcam, ab108337, 1:100 dilution), Osteocalcin (Abcam, ab93876, 1:100 dilution), CD146 (Abcam, ab75769, 1:100 dilution), CD140α (Abcam, ab96569, 1:100 dilution), CD200 (Abcam, ab203887, 1:200 dilution), Tartrate Resistant Acid Phosphatase (TRAP) (Abcam, ab185716, 1:50 dilution), and Cathepsin K (Abcam, ab19027, 1:200 dilution).
5
Comprehensive Tissue Staining Protocol
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All staining was performed on 2-µm-thick mouse tissue sections and 3-µm-thick human tissue sections. Hematoxylin and eosin (H&E) staining and MT staining were performed according to the standard protocols. The primary antibodies used for IHC/immunofluorescent (IF) staining were as follows: IL-6 (1:400, ab9324; Abcam), GFP (1:500, #598; Medical & Biological Laboratories), αSMA (1:400, ab5694; Abcam), NCAPG (1:200, 24563-1-AP; Proteintech), PTX3 (1:50, ab90806; Abcam), COMP (1:200, ab74524; Abcam), NCAM1 (1:100, ab133345; Abcam).
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