For ID1 pulldown experiments, Direct IP kit (Thermo Fisher Scientific) was used with polyclonal rabbit anti-ID1 antibodies (Abcam, #ab170511, #ab192303), or rabbit IgG isotype control (Thermo Fisher Scientific). RA ST (~0.5 cm3) homogenates were prepared in ice cold RIPA buffer supplemented with EDTA-free protease inhibitor cocktail (Thermo Fisher Scientific) using electric homogenizer and were centrifuged and filtered (45 μm) to collect the supernatant. For all IP experiments, manufacturer’s kit protocol was followed. Samples were eluted with low pH elution buffer supplied in the kit and prepared in Laemmli sample buffer for Western blotting; flow-throughs were retained for ELISA. All sera, SFs, and ST homogenates were incubated with polyclonal goat anti-human IgM (μ-chain specific)-conjugated Agarose (Sigma-Aldrich) at 4°C overnight for removal of RF prior to all immunoassays.
Goat anti human igm μ chain specific conjugated agarose
Manufactured by Merck Group
Goat anti-human IgM (μ-chain specific)-conjugated Agarose is a lab equipment product. It is an affinity matrix used for the purification or isolation of human IgM antibodies from biological samples.
Automatically generated - may contain errors
2 protocols using goat anti human igm μ chain specific conjugated agarose
1
ID1 Pulldown Experiments for RA Samples
2
Immunoprecipitation of ID-1 Protein
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For ID‐1 pulldown experiments, a Direct Immunoprecipitation kit (ThermoFisher Scientific) was used with polyclonal rabbit anti–ID‐1 antibodies (nos. ab170511 and ab192303; Abcam) or with rabbit IgG isotype control (ThermoFisher Scientific). RA ST (~0.5 cm3) homogenates were prepared in ice‐cold RIPA buffer supplemented with EDTA‐free protease inhibitor cocktail using an electric homogenizer and were centrifuged and filtered (45 μm) to collect the supernatant. For all immunoprecipitation experiments, manufacturers’ kit protocols were followed. Samples were eluted with low‐pH elution buffer supplied in the kit and prepared in Laemmli sample buffer for Western blotting; flowthroughs were retained for ELISA. All sera, SF, and ST homogenates were incubated with polyclonal goat anti‐human IgM (μ‐chain specific)–conjugated Agarose (Sigma‐Aldrich) at 4°C overnight for removal of RF prior to all immunoassays.
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