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3X FLAG Peptide Sale

(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) 카탈로그 번호: GP10149

합성 펩타이드 태그

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3X FLAG Peptide 화학 구조

Cas No.:402750-12-3

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Sample solution is provided at 25 µL, 10mM.

Product has been cited by 10 publications

Product Documents

품질 관리 및 안전 데이터 시트

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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.


[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.


FLAG-tag 시스템은 관심 단백질에 융합된 짧고 친수성인 8개의 아미노산 펩타이드를 이용합니다. FLAG 펩타이드는 항체 M1에 결합합니다. 결합이 칼슘 의존적인 방식2 또는 독립적인 방식3 인지 여부는 논란이 있습니다. 이 시스템의 단점은 모노클로날 항체 정제 매트릭스가 다른 것들보다 안정성이 낮다는 것입니다. 일반적으로 작은 태그는 특정한 모노클로날 항체로 감지됩니다.
FLAG 태그 검출을 개선하기 위해 3x FLAG 시스템이 개발되었습니다. 이 세 가닥의 FLAG 에피토프는 친수성으로, 22개의 아미노산으로 구성되어 있으며, 발현 융합 단백질을 최대 10 fmol까지 감지할 수 있습니다. Pyrococcus furiosus의 maltodextrin-binding protein에 FLAG 태그가 지원된 경우, 결정의 질은 untagged protein결정과 매우 유사합니다4.
마지막으로, enterokinase 처리를 통해 C-종단 다섯 개 아미노산 서열에 특이적인 FLAG 태그를 제거할 수 있습니다5.

1. Hopp TP, Prickett KS, Price VL, Libby RT, March CJ, Ceretti DP, Urdal DL, Conlon PJ (1988) 재조합 단백질 식별과 정제에 유용한 짧은 폴리펩타이드 마커 시퀀스. Bio/Technology 6:1204-1210.
2. Hopp TP, Gallis B, Prikett KS (1996) 칼슘 의존적인 모노클로날 항체의 금속 결합 특성. Mol Immunol 33:601-608.
3. Einhauer A, Jungbauer A (2000) FLAG 펩타이드를 대상으로 한 모노클로날 항체 M1의 운동학 및 열역학적 특성. 단백질 분리 국제 심포지엄(ISPPP), 슬로베니아 루블랴나에서 개최된 제20회(2000년 11월 5일-8일).
4. Bucher MH, Evdokimov AG,Waugh DS (2002) Pyrococcus furiosus maltodextrin-binding protein 결정화에 대한 짧은 친화력 태그의 차이점 영향.Biol Cryst58:392-397.
5.Maroux S., Baratti J., Desnuelle P. (1971) 돼지 엔테로키나제의 정제 및 특이성. J Biol Chem 246:5031-5039.

화학적 특성

Cas No. 402750-12-3 SDF
동의어 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
Chemical Name 3X FLAG Peptide
Formula C120H169N31O49S M.Wt 2861.87
용해도 ≥143.1mg/mL in DMSO, <14.35mg/mL in EtOH, ≥143.4mg/mL in Water with gentle warming Storage Desiccate at -20°C
일반적인 팁 높은 용해도를 얻기 위해 튜브를 37℃에서 데운 후 초음파 욕조에서 잠시 흔들어 주십시오. 원액은 -20℃ 이하에서 수개월간 보관할 수 있습니다.
배송 조건 평가 샘플 솔루션: 블루아이스와 함께 배송합니다. 다른 모든 가능한 크기: 요청 시 RT 또는 블루아이스와 함께 배송합니다.

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