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3X FLAG Peptide セール (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)

カタログ番号GP10149

合成ペプチドタグ

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3X FLAG Peptide 化学構造

Cas No.: 402750-12-3

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5mg
$65.00
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25mg
$264.00
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100mg
$740.00
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500mg
$2,218.00
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1g
$3,548.00
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Sample solution is provided at 25 µL, 10mM.

Product has been cited by 12 publications

Product Documents

Quality Control & SDS

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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.

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.

Background

FLAGタグシステムは、興味のあるタンパク質に融合された短い親水性8アミノ酸ペプチドを利用します。FLAGペプチドは抗体M1に結合します。結合がカルシウム依存的2または非依存的3であるかどうかは議論があります。このシステムの欠点は、モノクローナル抗体精製マトリックスが他のものほど安定していないことです。一般的に、小さなタグは特異的なモノクローナル抗体で検出することができます。
FLAGタグの検出を改善するために、3x FLAGシステムが開発されました。この三連続FLAGエピトープは親水性で22アミノ酸長であり、発現した融合タンパク質を10 fmolまで検出することができます。Pyrococcus furiosusのFLAG付きマルトデキストリン結合タンパク質が結晶化され4、その結晶品質は未修飾タンパク質のそれと非常に似ていました。
最後に、enterokinase処理により5つのC末端アミノ酸に特異的なFLAGタグを除去することができます。

参考文献:
1. Hopp TP、Prickett KS、Price VL、Libby RT、March CJ、Ceretti DP、Urdal DL、Conlon PJ(1988)「再生タンパク質の同定と精製に有用な短いポリペプチドマーカー配列」Bio/Technology 6:1204-1210。
2. Hopp TP、Gallis B、Prikett KS(1996)「カルシウム依存性モノクローナル抗体の金属結合特性」Mol Immunol 33:601-608。
3. Einhauer A, Jungbauer A (2000) 「FLAGペプチドに対するモノクローナル抗体M1の運動学的および熱力学的特性」20th International symposium on the separation of proteins, peptides, and polynucleotides (ISPPP). Lublijana, Slovenia, November 5-8, 2000.
4. Bucher MH, Evdokimov AG, Waugh DS (2002) 「Pyrococcus furiosus maltodextrin-binding proteinの結晶化に対する短い親和タグの異なる効果」Biol Cryst 58:392-397。
5. Maroux S, Baratti J, Desnuelle P (1971) 「豚腸エンテロキナーゼの精製と特異性」J Biol Chem 246:5031-5039。

Chemical Properties

Cas No. 402750-12-3 SDF
同義語 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
Canonical SMILES CCC(C)C(C(=O)NC(CC(=O)O)C(=O)NC(CC1=CC=C(C=C1)O)C(=O)NC(CCCCN)C(=O)NC(CC(=O)O)C(=O)NC(CC(=O)O)C(=O)NC(CC(=O)O)C(=O)NC(CC(=O)O)C(=O)NC(CCCCN)C(=O)O)NC(=O)C(CC(=O)O)NC(=O)C(CC2=CNC=N2)NC(=O)C(CC(=O)O)NC(=O)C(CCCCN)NC(=O)C(CC3=CC=C(C=C3)O)NC(=O)C(CC(=O)O)NC(
Formula C120H169N31O49S M.Wt 2861.87
溶解度 ≥143.1mg/mL in DMSO, <14.35mg/mL in EtOH, ≥143.4mg/mL in Water with gentle warming Storage Desiccate at -20°C
General tips Please select the appropriate solvent to prepare the stock solution according to the solubility of the product in different solvents; once the solution is prepared, please store it in separate packages to avoid product failure caused by repeated freezing and thawing.Storage method and period of the stock solution: When stored at -80°C, please use it within 6 months; when stored at -20°C, please use it within 1 month.
To increase solubility, heat the tube to 37°C and then oscillate in an ultrasonic bath for some time.
Shipping Condition Evaluation sample solution: shipped with blue ice. All other sizes available: with RT, or with Blue Ice upon request.

Complete Stock Solution Preparation Table

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1 mg 5 mg 10 mg
1 mM 0.3494 mL 1.7471 mL 3.4942 mL
5 mM 0.0699 mL 0.3494 mL 0.6988 mL
10 mM 0.0349 mL 0.1747 mL 0.3494 mL
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    3X FLAG Peptide- GlpBio

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