Immunoprecipitation and isolation of deiminated proteins in serum and serum-derived EVs was carried out using the Catch and Release®v2.0 immunoprecipitation kit (Merck, UK) in conjunction with the F95 pan-deimination antibody (MABN328, Merck), which has been developed against a deca-citrullinated peptide and specifically detects proteins modified by citrullination [252 (link)]. Bovine serum pools of the three individual animals (3 × 25 μL) were used for F95-enrichment from whole serum, while for EVs, total protein was first extracted from the pool of EVs derived from 3 animals (EV pellets derived from 100 μL serum per animal), using RIPA+ buffer (Sigma, UK). Following application of RIPA+ buffer, the EVs were incubated on ice for 2 h followed by centrifugation at 16,000× g for 30 min to collect the protein containing supernatant. Thereafter, immunoprecipitation (F95-enrichment) was carried out overnight on a rotating platform at 4 °C. The F95 bound proteins were eluted using denaturing elution buffer (Merck), according to the manufacturer’s instructions (Merck) and diluted 1:1 in Laemmli sample buffer. The F95-enriched eluates from whole serum and serum -EVs were then analyzed by SDS-PAGE, followed by silver staining, Western blotting or LC-MS/MS.
Denaturing elution buffer
Manufactured by Merck Group
Sourced in United Kingdom
Denaturing elution buffer is a laboratory buffer solution used in the elution step of protein purification processes. Its core function is to facilitate the release of target proteins from affinity chromatography resins by disrupting protein-ligand interactions through the use of chaotropic agents or changes in pH, ionic strength, or temperature.
Automatically generated - may contain errors
Lab products found in correlation
Catch and release v2.0 immunoprecipitation kit, by Merck Group (2 mentions)
Mabn328, by Merck Group (2 mentions)
Ripa buffer, by Merck Group (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
F95 pan deimination antibody, by Merck Group (1 mentions)
Catch and release immunoprecipitation kit, by Merck Group (1 mentions)
3 protocols using denaturing elution buffer
1
Deiminated Protein Enrichment from Serum and EVs
2
Deiminated Protein Enrichment and Analysis
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Immunoprecipitation and isolation of deiminated proteins in serum was carried out using the Catch and Release®v2.0 immunoprecipitation kit (Merck, U.K.) in conjunction with the F95 pandeimination antibody (MABN328, Merck), which has been developed against a deca-citrullinated peptide and specifically detects proteins modified by citrullination (Nicholas and Whitaker, 2002) .
Horseshoe crab serum pools of three individual animals (3 x 50 μl) were used for F95-enrichment.
Immunoprecipitation (F95-enrichment) was carried out overnight at 4 °C on a rotating platform. The F95 bound proteins were thereafter eluted using denaturing elution buffer (Merck), according to the manufacturer's instructions (Merck), diluted 1:1 in 2xLaemmli sample buffer (BioRad, UK) and kept frozen at -20 °C until further analysis by SDS-PAGE, followed by silver staining or western blotting, or by in-gel digestion followed by LC-MS/MS analysis.
Horseshoe crab serum pools of three individual animals (3 x 50 μl) were used for F95-enrichment.
Immunoprecipitation (F95-enrichment) was carried out overnight at 4 °C on a rotating platform. The F95 bound proteins were thereafter eluted using denaturing elution buffer (Merck), according to the manufacturer's instructions (Merck), diluted 1:1 in 2xLaemmli sample buffer (BioRad, UK) and kept frozen at -20 °C until further analysis by SDS-PAGE, followed by silver staining or western blotting, or by in-gel digestion followed by LC-MS/MS analysis.
3
Deiminated Protein Enrichment in Cetacean Sera
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For isolation of deiminated proteins from whale and orca sera, the F95 pan-deimination antibody (MABN328, Merck), which is developed against a deca-citrullinated peptide and specifically detects proteins modified by citrullination (Nicholas and Whitaker, 2002) , was used in conjunction with the Catch and Release immunoprecipitation kit (Merck, U.K.). According to the manufacturer's instructions (Merck), 50 μl of serum was used per sample for F95 enrichment and immunoprecipitation was carried out overnight on a rotating platform at 4 °C. The F95 bound proteins were eluted using denaturing elution buffer (according to the manufacturer's instructions, Merck) and the F95 enriched eluates were thereafter analysed both by Western blotting and by liquid chromatography mass spectrometry (LC-MS/MS) for identification of deiminated protein targets (Cambridge Proteomics, U.K.). For LC-MS/MS, peak files were submitted to in-house Mascot (Matrix Science, Cambridge Proteomics) using the following databases for identification of species-
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