Samples for co-immunoprecipitation and mass spectrometry were prepared using Thermo Scientific™ Pierce™ Co-Immunoprecipitation Kit according to manufacturer's protocol. For mass spectrometry, samples were precipitated with anti-FAP polyclonal antibody (R&D systems, Cat # AF3715) and then sent to UF ICBR for analysis on TSQ Altis™ Triple Quadrupole Mass Spectrometer (Thermo Fisher Scientific). For co-immunoprecipitation, samples were either precipitated with anti-FAP polyclonal antibody or goat anti-PRKDC polyclonal antibody (Abcam, Cat # ab168854).for 2 h and further with TrueBlot™ anti-Rabbit Ig Beads or TrueBlot™ anti-sheep Ig Beads ((Rockland, Limerick, PA). After several washes, beads were boiled and subjected to western blot analysis.
Trueblot anti rabbit ig beads
Manufactured by Rockland Immunochemicals
Sourced in Ireland
TrueBlot™ anti-Rabbit Ig Beads are agarose beads coated with an antibody that specifically binds to the Fc region of rabbit immunoglobulins (Ig). These beads are designed for the efficient capture and purification of rabbit antibodies from complex samples.
Automatically generated - may contain errors
2 protocols using trueblot anti rabbit ig beads
1
Co-immunoprecipitation and Mass Spectrometry Analysis
2
Immunoprecipitation of RUNX2 and STAT1
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Cell extracts were harvested in a Pierce IP Lysis Buffer® (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol). 500 μg cell extracts were incubated with 1 μg anti-RUNX2 (M-70, Santa Cruz, Catalog No.sc-10758), or STAT1 (Cell signaling, Catalog No.9172S) at 4° C overnight. Immune complexes were recovered with True Blot® anti-rabbit Ig beads (Rockland, Catalog No.00-8800-25) and subjected to SDS-PAGE.
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