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  • 3X FLAG Peptide
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3X FLAG Peptide

3×FLAG Peptide是一个合成的含有三个重复DYKXXD氨基酸序列的多肽,常用于竞争性结合Anti-Flag抗体。

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3X FLAG Peptide的二维码
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  • 货号: ajcp4724
  • CAS: 402750-12-3
  • 别名: 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
  • 分子式: C120H169N31O49S
  • 分子量: 2861.87
  • 纯度: ＞98%
  • 溶解度: ≥100mg/mL in Water (Ultrasonic assisted dissolution; Dissolve under acidic conditions (HCl adjustment, pH≈2))
  • 储存: Desiccate at -20°C
  • 库存: 现货

Background

3X FLAG Peptide is a synthetic polypeptide containing three repeated DYKXXD amino acid sequences, often used to competitively bind Anti-Flag antibodies.3X Flag Peptide is mainly used for protein identification and purification, such as elution of Flag fusion expression proteins bound to Anti-Flag antibodies during immunoprecipitation[1-5].

参考文献:
[1]. Hopp, T., Prickett, K., et al. A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification. Nat Biotechnol 6, 1204-1210 (1988). https://doi.org/10.1038/nbt1088-1204
[2]. Einhauer A, Jungbauer A. Affinity of the monoclonal antibody M1 directed against the FLAG peptide. J Chromatogr A. 2001 Jun 29;921(1):25-30. doi: 10.1016/s0021-9673(01)00831-7. PMID: 11461009.
[3].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.
[4].Gao J, Huang G, et al.PROTEIN S-ACYL TRANSFERASE 13/16 modulate disease resistance by S-acylation of the nucleotide binding, leucine-rich repeat protein R5L1 in Arabidopsis. J Integr Plant Biol. 2022 Sep;64(9):1789-1802. doi: 10.1111/jipb.13324. Epub 2022 Jul 28. PMID: 35778928.
[5]. Lian H, Jiang K, et al.The Salmonella effector protein SopD targets Rab8 to positively and negatively modulate the inflammatory response. Nat Microbiol. 2021 May;6(5):658-671. doi: 10.1038/s41564-021-00866-3. Epub 2021 Feb 18. PMID: 33603205; PMCID: PMC8085087.

3×FLAG Peptide是一个合成的含有三个重复DYKXXD氨基酸序列的多肽,常用于竞争性结合Anti-Flag抗体。3× Flag Peptide 主要用于蛋白鉴定与纯化,比如可以在免疫沉淀时洗脱与Anti-Flag抗体结合的Flag融合表达蛋白的多肽[1-5]。

Protocol

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.

参考文献:

[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.
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