For identification of proteins interacting with GAG, the BMDMs were lysed in NP40 buffer (50 mM HEPES, 150 mM NaCl, 1% NP40, 50 mM EDTA, pH 7.4 ± 0.2) and centrifuged for 10 min at 10,000 × g to remove cell debris. Cell lysate and GAGs (GAG, Ac-GAG, d-GAG) or β-glucan were incubated for 2 h with rotation at room temperature with or without 1.2 M NaCl (BP358, Fisher). The samples were washed 6 times with NP40 buffer after 10 min of 10,000 × g centrifugation at 4°C. After washes, polysaccharides pulled down were eluted in sample buffer and used for immunoblotting analysis or mass spectrometry analysis on the Q-Exactive mass spectrometer as describe previously17 (link).
Protocol detail
Identifying Protein-GAG Interactions
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Buffer
Cell
Centrifugation
Edta
Hepes
Immunoblotting analysis
Mass spectrometry
Nacl
Polysaccharides
Proteins
β glucan
Corresponding Organization :
Other organizations : St. Jude Children's Research Hospital, Laboratoire de Biologie, Bioingéniérie et Bioimagerie ostéoarticulaire, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Institut thématique Immunologie, inflammation, infectiologie et microbiologie, Architecture et Fonction des Macromolécules Biologiques, King Abdulaziz University
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Variable analysis
independent variables
- GAGs (GAG, Ac-GAG, d-GAG) or β-glucan
- 1.2 M NaCl (BP358, Fisher)
- Proteins interacting with GAG
- BMDM cell lysate
- NP40 buffer (50 mM HEPES, 150 mM NaCl, 1% NP40, 50 mM EDTA, pH 7.4 ± 0.2)
- Incubation time (2 h)
- Incubation temperature (room temperature)
- Centrifugation time (10 min)
- Centrifugation speed (10,000 × g)
- Number of washes (6 times)
- No GAG or β-glucan control (lysate incubated without polysaccharides)
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