Tandem affinity purification and identification of ILDR2-interacting proteins
This protocol is extracted from research article:
ILDR2 stabilization is regulated by its interaction with GRP78
Sci Rep, Apr 16, 2021; DOI: 10.1038/s41598-021-87884-7

MIN6 cells infected with adenovirus vector encoding tandem affinity purification-tagged ILDR2 proteins were collected and suspended with lysis buffer (10 mm Tris–HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, and protease inhibitor cocktail (Roche)). Lysates were centrifuged at 15 000 × g at 4 °C for 20 min, and the resulting supernatants were loaded into Anti-FLAG M2 agarose affinity gel (Sigma-Aldrich) and rotated for 60 min at 4 °C. The resin was washed five times with wash buffer (10 mm Tris–HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.2% NP-40, and protease inhibitor cocktail), and bound proteins were eluted with 150 μg/ml 3xFLAG peptide in binding buffer (50 mM NaH2PO4 and 300 mM NaCl) for Ni–NTA purification system. Eluted fractions were next loaded into Ni–NTA agarose (Qiagen) and rotated for 60 min at 4 °C. The resin was washed five times with binding buffer and then the ILDR2 protein complexes were eluted with in elution buffer (50 mM NaH2PO4, 300 mM NaCl, and 250 mm imidazole). The ILDR2 protein complexes were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane. The protein bands of interest were excised from the PVDF membrane and digested with trypsin. The trypsinized peptides were then treated with 50% acetonitrile containing 0.1% trifluoroacetic acid and desalted with a Ziptip C18. The peptides were analyzed by matrix-assisted laser desorption-ionization/time of flight mass spectrometry (MALDI/TOF–MS), Autoflex (Bruker Daltonics). Detected masses and the sequences of the peptides were subjected to database searches using Mascot software (Matrix Science)11.

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