3X FLAG peptide

3X FLAG peptides are FLAG-tagged peptides containing three repeats of the Asp-Tyr-Lys-Xaa-Xaa-Asp motif. 3X FLAG peptide can be used for protein separation and purification, and competitive elution with target proteins. In Vitro:1. Purification of FLAG tagged proteins through 3X FLAG peptide competition^[2]
(1) Plasmid transfection: Transfect plasmids of mIRS-1, hIRS-1, or their mutants labeled with FLAG into 293 T cells for 2 days.
(2) Cell collection: Cells were harvested by centrifugation at 14000 rpm for 10 minutes at 4 °C, and lysed with HEPES buffer (150 mM NaCl, 50 mM HEPES, pH 7.4, 5 mM EDTA, 1% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100, 0.2% sodium fluoride, 0.1% sodium orthovanadate, and protease inhibitor mixture).
(3) Using Anti-FLAG magnetic beads for protein separation and purification: Add Anti-Flag magnetic beads (HY-K0207)/Anti-c-Myc magnetic beads (1 μm) (HY-K0207A) to the cell lysate, and incubate at room temperature for 2 hours or at 4 °C overnight. Then wash with a washing buffer and elute with a buffer containing 1 mg/mL 3X FLAG. Finally, use a gel filtration column for further purification of the supernatant, which is equilibrated with the storage buffer. All purified proteins are concentrated using centrifugal filtration and aliquoted for storage at -80 °C.
(4) Protein quantification and validation: The purified protein was quantified using an ND-2000 NanoDrop spectrophotometer with OD 280 and validated by Coomassie Brilliant Blue staining.
2. Co-IP and co-IP-MS analysis using the 3X FLAG peptide^[3]
(1) Centrifuge the cell lysate at 13300 rpm for 15 minutes at 4 °C to remove intact cells.
(2) The supernatant was incubated overnight with control immunoglobulin G (IgG) or primary antibody in immunoprecipitation buffer (150 mM NaCl, 20 mM HEPES, pH 7.4, 1% Triton X-100, 12.5 mM β - glycerophosphate, 1.5 mM MgCl₂, 2 mM EGTA, containing phosphatase and protease inhibitors), and then incubated with 20 μL resuspended A/G protein beads at 4 °C for 2 hours to precipitate bound proteins.
(3) For continuous immunoprecipitation, incubate 3X FLAG eluent solution (final concentration 150 μg/mL) with 5 times the volume of gel at 4 °C for 2 hours, elute the bound protein from beads, followed by Western blotting.
(4) Alternatively, the samples were run on SDS-PAGE gel and then dyed (Bio Rad). Subsequently, cut off the tape, decolorize, digest with trypsin, and perform MS analysis to determine individual proteins using liquid chromatography-MS.