Synovial fluid (stored at − 80 °C) was used to quantify total protein using the Bradford colorimetric method [30 (link)]. For this, 50 μg of total protein were separated on 12% SDS-PAGE gels at 100 V for 2 h and transferred onto PVDF membranes at 200 mA for 2 h. Membranes were then blocked in Tris-buffered saline with Tween-20 (TBS-T) buffer (10 mM Tris–HCl, 68 mM NaCl, and 0.1% Tween-20) and 5% non-fat dry milk for 2 h at RT and washed with TBS-T. The primary antibodies used were: histone H3 (citrulline R2 + R8 + R17; H3cit) antibody (#ab5103, Abcam, UK) and serpin B1 antibody (#ARP52417_P050, Aviva Systems Biology, CA. USA) incubated at 4 °C in constant agitation overnight. Thereafter secondary antibodies [anti-rabbit IRDye 800 and 680 (Li-cor Biosciences, USA)] were used. Analysis of membranes was performed by using a LI-COR OdysseyFc system (LI-COR Biosciences, NE, USA) and densitometry was quantified using the Image 1.51j8 software.
H3cit antibody
Manufactured by Abcam
Sourced in United Kingdom
H3cit) antibody is a laboratory research tool that specifically binds to histone H3 proteins with citrullinated arginine 2. This antibody can be used to detect and study the presence and distribution of this post-translational modification in various biological samples.
Automatically generated - may contain errors
2 protocols using h3cit antibody
1
Quantification of Synovial Fluid Proteins
2
Neutrophil Calcium Ionophore Stimulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Primary mouse neutrophils in RPMI-1640 medium containing 25 mmol/L HEPES were seeded onto each well of an eight-well chamber slide. Twenty minutes later, attached cells were incubated with 5 μmol/L calcium ionophore A23187 for 2 h before being fixed and stained with H3Cit antibody (1:100 dilution; Abcam) and DAPI (Thermo Fisher Scientific). Images were taken with Carl Zeiss LSM 710 confocal microscopy and analyzed with ImageJ.
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