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3X FLAG Peptide Verkauf (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)

Katalog-Nr.GP10149

Synthetisches Peptid-Tag

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3X FLAG Peptide Chemische Struktur

Cas No.: 402750-12-3

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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

Das FLAG-Tag-System verwendet ein kurzes, hydrophiles 8-Aminosäure-Peptid, das mit dem Protein von Interesse fusioniert wird. Das FLAG-Peptid bindet an den Antikörper M1. Ob die Bindung calciumabhängig oder -unabhängig ist, bleibt umstritten. Ein Nachteil des Systems besteht darin, dass die Monoklonalantikörper-Reinigungsmatrix nicht so stabil ist wie andere. Im Allgemeinen können kleine Tags mit spezifischen monoklonalen Antikörpern nachgewiesen werden.
Um die Erkennung des FLAG-Tags zu verbessern, wurde das 3x-FLAG-System entwickelt. Dieses dreifache Flag-Epitop ist hydrophil, 22 Aminosäuren lang und kann bis zu 10 fmol exprimiertes Fusionsprotein erkennen. Das mit einem FLAG-Tag versehene Malto-Dextrin-bindende Protein von Pyrococcus furiosus wurde kristallisiert und die Qualität der Kristalle war sehr ähnlich wie bei Kristallen des unmarkierten Proteins.
Schließlich kann das FLAG-Tag durch Behandlung mit Enterokinase entfernt werden, die spezifisch für die fünf C-terminalen Aminosäuren der Peptidsequenz ist.

Referenzen:
1. Hopp TP, Prickett KS, Price VL, Libby RT, March CJ, Ceretti DP, Urdal DL, Conlon PJ (1988) Eine kurze Polypeptid-Markersequenz zur Identifikation und Reinigung von rekombinanten Proteinen. Bio/Technology 6:1204每1210.
2. Hopp TP, Gallis B, Prikett KS (1996) Metallbindende Eigenschaften eines calciumabhängigen monoklonalen Antikörpers. Mol Immunol 33:601每608.
3. Einhauer A., Jungbauer A (2000) Kinetik und thermodynamische Eigenschaften des monoklonalen Antikörpers M1 gegen das FLAG-Peptid. 20th International symposium on the separation of proteins, peptides and polynucleotides (ISPPP). Lublijana Slowenien vom 5.-8.November 2000.
4. Bucher MH., Evdokimov AG., Waugh DS.(2002) Unterschiedliche Auswirkungen kurzer Affinitätsmarkierungen auf die Kristallisation des Pyrococcus furiosus Maltodextrin-bindenden Proteins.Biol Cryst58:392每397.
5.Maroux S., Baratti J., Desnuelle P.(1971) Reinigung und Spezifität der Schweine-Enterokinase.J Biol Chem246:5031每5039.

Chemical Properties

Cas No. 402750-12-3 SDF
Überlieferungen 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
Löslichkeit ≥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

Prepare stock solution
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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