3X FLAG Peptide (Synonyms: H-Met-Asp-Tyr-Lys-Asp-His-Asp-Gly-Asp-Tyr-Lys-Asp-His-Asp-Ile-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-OH)

Protocol for Protein expression and purification by 3×FLAG peptide ^[1]:

1. The plasmids of FLAG-tagged genes were transfected into 293T cells for 2 days.
2. The Cells were harvested by centrifugation at 14,000 rpm for 10 min at 4℃ and lysed with HEPES buffer (150 mM sodium chloride, 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 cocktail).
3. Anti-FLAG M2 gel beads were then added and incubated on a rotary shaker at 4 C for 2 h. M2 gel beads were harvested by centrifugation at 3,000 rpm for 1 min and washed four times in HEPES buffer.
4. The FLAG-tagged proteins were purified by the competition of 3×FLAG peptide. Briefly, M2 beads were resuspended with 1.5 mg/mL 3×FLAG peptide buffer and incubated at 4℃ for 2 h.
5. After centrifugation at 5,000 rpm for 1 min, the supernatant further purified by gel filtration using a SuperdexTM 200 increase column equilibrated in store buffer (50 mM Tris-HCl, 37 mM NaCI, 1 mM EDTA, 5 mM DTT).
6. All the purified proteins were concentrated by centrifugal filtration and stored in aliquots at 80℃.
7. The purified protein was quantified using a ND-2000 NanoDrop spectrophotometer with OD 280 and verified by Coomassie staining.

This protocol only provides a guideline, and should be modified according to your specific needs.

Protocol for Co-IP and co-IP–MS analyses using 3×FLAG peptide ^[2]:

1. Cell lysates were centrifuged at 13,300 r.p.m. for 15 min at 4℃ to remove intact cells.
2. The supernatant was either incubated with control immunoglobulin G (IgG) or primary antibody overnight in IP buffer (150 mM NaCl, 20 mM HEPES at pH 7.4, 1% Triton X-100, 12.5 mM β-glycerophosphate, 1.5 mM MgCl₂, 2 mM EGTA with phosphatase and protease inhibitors), followed by incubation with 20 μl of resuspended volume of Protein A/G beads for 2 h at 4℃ to pull down bound proteins.
3. For sequential IP, bound protein was eluted from the beads by incubation with 5 packed gel volumes of 3×FLAG peptide elution solution (150 ng/ml final concentration) at 4℃ for 2 h and then immunoprecipitated with the second antibody overnight at 4℃.
4. Beads were centrifuged at 1,000g for 5 min at 4℃ to remove the supernatant, washed 4 times with the IP buffer and boiled for 20 min at 95℃. Samples were run on SDS PAGE gel, followed by Coomassie Brilliant Blue R-250 staining.
5. Afterwards, gel bands were excised, destained, trypsinized and subjected to MS analysis to identify individual proteins using liquid chromatography MS.

This protocol only provides a guideline, and should be modified according to your specific needs.

References:

[1].  Gao XK, Rao XS, et al. Phase separation of insulin receptor substrate 1 drives the formation of insulin/IGF-1 signalosomes. Cell Discov. 2022 Jun 28;8(1):60. doi: 10.1038/s41421-022-00426-x. PMID: 35764611; PMCID: PMC9240053.

[2].  Cong M, Wang Y, et al. MTSS1 suppresses mammary tumor-initiating cells by enhancing RBCK1-mediated p65 ubiquitination. Nat Cancer. 2020 Feb;1(2):222-234. doi: 10.1038/s43018-019-0021-y. Epub 2020 Jan 20. PMID: 35122005.