الصفحة الرئيسية>>Peptides>> Tag Peptides>>3X FLAG Peptide

3X FLAG Peptide بيع (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)

رقم الكتالوجGP10149

علامة الببتيد الاصطناعية

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3X FLAG Peptide التركيب الكيميائي

Cas No.: 402750-12-3

الحجم السعر المخزون الكميّة
5mg
65٫00
متوفر
25mg
264٫00
متوفر
100mg
740٫00
متوفر
500mg
2218٫00
متوفر
1g
3548٫00
متوفر

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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

يستخدم نظام FLAG-tag ببتيد قصير وهيدروفيليكي يحتوي على 8 أحماض أمينية ملتصقة بالبروتين المراد دراسته. يرتبط الببتيد FLAG بالأجسام المضادة M1، ولا يزال الجدل قائمًا حول ما إذا كان الارتباط في طريقة تعتمد على الكالسيوم2 أو لا3. يعود سلبية هذه الطريقة إلى عدم استقرار مصفى التنقية من الأجسام المضادة المونوكلونية بشكل جدير بالثقة كغيره من المصافى. وعلى العموم، يُستطَّلَعُ التَّاغاتُ الصِّغيرَةُ باستخدامِ أجسامٍ مضادَّةٍ خاصَّة.
تم تطوير نظام 3x FLAG لزيادة اكتشاف التاغ FLAG. هذه التاغ ثلاثية التسلسل (tandem) Flag epitope هى هذروفيلية، طولها 22 حامضًأ اورث-أحادى، وإن شاء رابط حزمة البروتين المراد دراسته يمكن اكتشاف ما يصل إلى 10 فمول من البروتينات المدمجة. تبلور بروتين رابط FLAG-tagged maltodextrin-binding protein of Pyrococcus furiosus4 وكان جودة التبلور شديد التشابه مع تبلور البروتين غير المرادف.
أخيرًا، يُمْكِنُ إزالَةُ عَلامَةِ FLAG-tag بالعلاج بإنْتِرو-أُكِيْناس، وهى خاصّةٌ لآخر خمس حامضات أحادية C-terminal amino acids في سلسلة الببتيد5.

المراجع:
1. هوب تي بي، بريكت كيه إس، برايس في ال، ليبي آر تي، مارش س جاى، سيرتى دى بى، أوردال ديل و كونلون پ جاى (1988) علامة قصيرة من الببتيدات المفيدة لتحديد وتنقية البروتينات المستحثة. Bio/Technology 6:1204每1210.
2. هوب تی بی، جالیس بـــــروكِّەت کێ اس (1996) خصائص رابطة المعادن لأجسام مضادة أحادية الكالس يؤثر علیها. Mol Immunol 33:601-608.
3. إینهاور أ, يانغ باور A (2000) خصائص حركية وحرارية للجزء المضاد M1 التابع للپپتید FLAG .20th International symposium on the separation of proteins, peptides, and polynucleotides(ISPPP). Lublijana, Slovenia, November 5每8, 2000.
4. Bucher MH,Evdokimov AG,Waugh DS(2002) التأثيرات المختلفة للاشارات القصيرة على التبلور من Pyrococcus furiosus maltodextrin-binding protein. Biol Cryst 58:392-397.
5. Maroux S, Baratti J, Desnuelle P (1971) تنقية وتحديد الخصوصية للإنتروكيناز الخنزيري. J Biol Chem 246:5031-5039.

Chemical Properties

Cas No. 402750-12-3 SDF
المرادفات 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
الذوبان ≥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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