400-998-5282
专注多肽 服务科研
400-998-5282
专注多肽 服务科研
3Xflag能够使可免疫原性和与抗体的亲和性得到增强。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。
编号：184546
CAS号：402750-12-3
单字母：H2N-MDYKDHDGDYKDHDIDYKDDDDK-OH
| 实验参考方法: | Protocol for Protein expression and purification by 3×FLAG peptide [1]: The plasmids of FLAG-tagged genes were transfected into 293T cells for 2 days. 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). 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. 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. 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). All the purified proteins were concentrated by centrifugal filtration and stored in aliquots at 80℃. 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]: Cell lysates were centrifuged at 13,300 r.p.m. for 15 min at 4℃ to remove intact cells. 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. 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℃. 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. 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. |
3X FLAG peptide TFA 是一种合成的多肽，包含三个重复的 Asp-Tyr-Lys-Xaa-Xaa-Asp 基序。
Flag多肽为编码8个氨基酸的亲水性多肽（DYKDDDDK），可用于检测特异性anti-Flag单抗，为提高检测灵敏性，现研发出3×Flag重复多肽（3×Flag-tag Peptide），该3×Flag重复多肽含23个氨基酸，可以检测高达10fmol融合蛋白。3×Flag重复多肽已广泛用于纯化Flag融合蛋白，竞争性结合Anti-Flag单抗。
3× FLAG标签系统大幅度提高了FLAG标签的检测能力。三串联的FLAG表位是亲水性的，22个氨基酸的长链，它最多可以检测10 fmol的融合蛋白。FLAG标记的Pyrococcus furiosus的麦芽糊精结合蛋白已经获得了晶体，晶体结果显示FLAG标签对蛋白晶体质量没有多大影响。
FLAG标签可以用肠激酶法处理去除，肠激酶能够特异性的作用于C端的5个氨基酸多肽序列。
用途：3-Flag peptide被广泛用作一种温和的纯化试剂，用于Flag表位标记重组蛋白的纯化。尽管在缺乏钙离子时，其亲和柱会释放单价标记蛋白，但是抗体仍保留对Flag序列的亲和性，即使在无金属存在的条件下，因此不能用来进行金属敏感的ELISA实验。当Flag标记蛋白结合在ELISA板或印迹过滤器上时，抗体仍然与多价表面包被抗原结合。抗原多价性提高了Flag抗体的亲和性，使得ELISA反应不依赖于钙离子。然而，当抗体本身是单价的，即通过蛋白水解变成Fab，ELISA反应即变为钙依赖的。这种金属依赖的ELISA实验被用于研究抗体对金属的需求。在二价金属中，比钙半径大或比钙半径小的金属其结合均逐渐减少。几种更小的金属，比如镍，充当结合反应的抑制剂。重金属如镉、镧和钐可以与抗体结合。由于对该抗体与重组Flag融合蛋白共结晶的兴趣，其与重金属结合的能力就变得非常有意义。
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]。
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].
References:
[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.
3x DYKDDDDK peptide, also called 3x FLAG peptide, is a 23 aa length peptide used for the competitive elution of DYKDDDDK-tag fusion proteins (with excess of free 3x DYKDDDDK peptide) under non-denaturing conditions, especially when a DYKDDDDK-tagged protein is sensitive to low pH.\n\n\nInvolvement in COVID-19 disease\n\nSome recombinant proteins, such as Spike glycoprotein and ACE2 are frequently produced using the FLAG-tag system. In order to elute such proteins purified by affinity, sb-PEPTIDE provides DYKDDDDK peptide and 3x DYKDDDDK peptide.\n\nTo help your research for COVID-19 disease, we can also provide you with premium quality peptide libraries containing SARS-CoV-2 proteins. Our expertise make us able to synthesize glycopeptides and phosphopeptides to be close from the real content of these peptides. For more information, please visit this link
专肽生物可以提供常见抗体制备、免疫学研究等生物科研需求的各类标签多肽（Tag peptide）以及蛋白底物产品，可用荧光标记(FITC、5-Fam)等。除常见抗体中抗原决定部位的标签多肽（tag: His | c-Myc | HA | FLAG | V5 | X-press | VSV tag | T7 tag | Snoop tag | E-tag | Sof tag 1 | Spy tag |等等），我们还提供各类定制标签肽的合成，并可提供荧光标记(FITC、5-Fam)等。同时，免费提供的MS和HPLC分析报告。
| DOI | 名称 | |
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| 10.21769/BioProtoc.3616 | Purification of Protein-complexes from the Cyanobacterium Synechocystis sp. PCC 6803 Using FLAG-affinity Chromatography | 下载 |
| 10.1016/0161-5890(96)00026-0 | Metal-binding properties of a calcium dependent monoclonal antibody. | 下载 |
| 10.1107/s0907444901021187 | Differential effects of short affinity tags on the crystallization of Pyrococcus furiosus maltodextrin-binding protein. | 下载 |
