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FLAG tag Peptide Sale

(Synonyms: H-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-OH) 目录号 : GP10150

FLAG tag Peptide is an 8-peptide (Asp-Tyr-lys-Asp-Asp-Asp-ASP-ASP-Lys) containing an intestinal kinase restriction site.

FLAG tag Peptide Chemical Structure

Cas No.:98849-88-8

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1mg
¥350.00
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5mg
¥630.00
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10mg
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25mg
¥1,673.00
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Sample solution is provided at 25 µL, 10mM.

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实验参考方法

Purification of ribonucleoprotein (RNP) complexes composed of specific cellular rna by FLAG peptide coupled antisense oligonucleotide (ASO) pull-down [1]:

Preparation Method

Preparation of FLAG tag Peptide-Conjugated Antisense Oligonucleotide

1.Mix 50 μL of 20 μM (1,000 pmol/50 μL) ice-cold oligonucleotide with 50 μL of freshly prepared cold 1 M NaIO4.

2.Incubate on ice for 10 min.

3.Add 1 mL of ice-cold 2 % LiClO4/acetone and vortex.

4.Incubate on ice for 10 min.

5.Centrifuge the tube at 20,000 g at 4 ℃ for 10 min.

6.Carefully aspirate the supernatant to waste and add 1 mL of cold acetone.

7.Centrifuge the tube at 20,000 g at 4℃ for 5 min.

8.During the centrifugation, prepare the FLAG-hydrazide solution.

9.Aspirate the supernatant to waste.

10.Dissolve the precipitated oligonucleotide in 12 μL of 0.1 M sodium acetate (pH 5.2).

11.Add 12 μL of 30 mM FLAG-peptide-hydrazide solution.

12.Incubate the tube at room temperature for 30 min.

13.Add 10 μL of 1 M NaCNBH3.

14.Incubate the tube at room temperature for 30 min.

15.Add 60 μL of ultrapure water and 10 μL of 3 M sodium acetate (pH 5.2), then vortex.

16.Add 250 μL of 100 % ethanol and vortex.

17.Incubate the tube at −80℃ for 30 min.

18.Centrifuge the tube at 15,000 g for 10 min.

19.Carefully aspirate the supernatant to waste and add 1 mL of 80 % ethanol.

20.Centrifuge the tube at 15,000 g for 5 min.

21.Carefully aspirate the supernatant to waste. Repeat the wash step.

22.Dissolve the oligonucleotide pellet in 50 μL of water.

23.Estimate the concentration of the FLAG-peptide-conjugated-oligonucleotide by measuring the absorbance at 260 nm and comparing with the absorbance of the original oligonucleotide solution.

References:

[1]. Adachi S, Natsume T. Purification of noncoding RNA and bound proteins using FLAG peptide-conjugated antisense-oligonucleotides. Methods Mol Biol. 2015;1262:265-74. doi: 10.1007/978-1-4939-2253-6_16. PMID: 25555587.

产品描述

FLAG tag Peptide is an 8-peptide (Asp-Tyr-lys-Asp-Asp-Asp-ASP-ASP-Lys) containing an intestinal kinase restriction site. FLAG Tag peptide fusion system is used in immunoaffinity chromatography for protein identification and purification. FLAG tag Peptide label peptides allow elution under non-denatured conditions[1,6]. The FLAG tag Peptide can be fused to either the N- or C-terminus of a given fusion protein.

The FLAG tag Peptide marker peptide fusion system comprises a unique and widely useful technique for protein identification and purification. When covalently attached to a solid support, the anti-Flag M1 antibody can be used for the rapid purification of FLAG tag Peptide fusion proteins in a mild, calcium-dependent affinity chromatography procedure. FLAG tag Peptide fusion proteins are typically purified to homogeneity in a single step, starting from a crude cell homogenate or supernatant, without ever exposing the protein to conditions other than physiological saline at pH 7.2 (with calcium or EDTA). The entire purification process can be carried out within several hours[2-3].

The N-terminal FLAG tag Peptide fusion protein can be purified by agarose affinity gel[5]. A short-cut protein purification procedure, where mechanical cell disintegration and affinity purification by immobilization of the anti-Flag M1 antibody on magnetic glass beads are combined. The modified glass beads were added to yeast cells and mixed on a laboratory vortex mixer. The magnetic glass beads capture the target protein, while the yeast cells are disrupted. By making contact with a stationary magnet, the magnetic glass beads can very efficiently be separated from yeast cell debris. Chelating agents, such as EDTA, were capable of eluting the FLAG tag Peptide fusion proteins from the magnetic glass beads[4].

FLAG 标签肽是一种包含肠激酶限制性位点的 8 肽 (Asp-Tyr-lys-Asp-Asp-Asp-ASP-ASP-Lys)。 FLAG Tag 肽融合系统用于免疫亲和层析,用于蛋白质鉴定和纯化。 FLAG 标签肽标签肽允许在非变性条件下洗脱[1,6]。 FLAG 标签肽可以融合到给定融合蛋白的 N 端或 C 端。

FLAG 标签肽标记肽融合系统包含一种独特且广泛有用的蛋白质鉴定和纯化技术。当共价连接到固体支持物上时,抗 Flag M1 抗体可用于在温和的钙依赖性亲和层析过程中快速纯化 FLAG 标签肽融合蛋白。 FLAG 标签肽融合蛋白通常从粗细胞匀浆或上清液开始一步纯化至均质,除了 pH 7.2 的生理盐水(含钙或 EDTA)外,从未将蛋白质暴露于其他条件。整个纯化过程可在数小时内完成[2-3]

N-terminal FLAG tag Peptide融合蛋白可以通过琼脂糖亲和凝胶纯化[5]。一种快捷的蛋白质纯化程序,其中结合了机械细胞分解和通过将抗 Flag M1 抗体固定在磁性玻璃珠上进行的亲和纯化。将改性玻璃珠添加到酵母细胞中并在实验室涡旋混合器上混合。磁性玻璃珠捕获目标蛋白,同时破坏酵母细胞。通过与固定磁铁接触,磁性玻璃珠可以非常有效地与酵母细胞碎片分离。 EDTA 等螯合剂能够从磁性玻璃珠上洗脱 FLAG 标签肽融合蛋白[4]

References:
[1]. Einhauer A, Jungbauer A. The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. J Biochem Biophys Methods. 2001 Oct 30;49(1-3):455-65. doi: 10.1016/s0165-022x(01)00213-5. PMID: 11694294.
[2]. Chiang CM, Roeder RG. Expression and purification of general transcription factors by FLAG epitope-tagging and peptide elution. Pept Res. 1993 Mar-Apr;6(2):62-4. PMID: 7683509.
[3]. Prickett KS, Amberg DC, Hopp TP. A calcium-dependent antibody for identification and purification of recombinant proteins. Biotechniques. 1989 Jun;7(6):580-9. PMID: 2698650.
[4]. Schuster M, Wasserbauer E, Ortner C, Graumann K, Jungbauer A, Hammerschmid F, Werner G. Short cut of protein purification by integration of cell-disrupture and affinity extraction. Bioseparation. 2000;9(2):59-67. doi: 10.1023/a:1008135913202. PMID: 10892539.
[5]. Hopp TP, Prickett KS, Price VL, et al. A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification. Bio/technology (Nature Publishing Company). 1988;6:1204-1210. DOI: 10.1038/nbt1088-1204.
[6].Li Y. Commonly used tag combinations for tandem affinity purification. Biotechnol Appl Biochem. 2010 Feb 15;55(2):73-83. doi: 10.1042/BA20090273. PMID: 20156193.

Chemical Properties

Cas No. 98849-88-8 SDF
别名 H-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-OH
化学名 FLAG Peptide
Canonical SMILES C1=CC(=CC=C1CC(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)N)O
分子式 C41H60N10O20 分子量 1012.97
溶解度 ≥ 50.6 mg/mL in DMSO, ≥ 210.6 mg/mL in Water, ≥ 34.03 mg/mL in EtOH 储存条件 Store at -20°C, protect from light, stored under nitrogen
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储备液的保存方式和期限:-80°C 储存时,请在 6 个月内使用,-20°C 储存时,请在 1 个月内使用。
为了提高溶解度,请将管子加热至37℃,然后在超声波浴中震荡一段时间。
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溶解性数据

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1 mg 5 mg 10 mg
1 mM 0.9872 mL 4.936 mL 9.872 mL
5 mM 0.1974 mL 0.9872 mL 1.9744 mL
10 mM 0.0987 mL 0.4936 mL 0.9872 mL
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Research Update

Effect of N-glycosylation on turnover and subcellular distribution of N-acetylgalactosaminyltransferase I and sialyltransferase II in neuroblastoma cells

Gangliosides are sialylated glycosphingolipids whose biosynthesis is catalyzed by a series of endoplasmic reticulum (ER)- and Golgi-resident glycosyltransferases. Protein expression, processing, and subcellular localization of the key regulatory enzymes for ganglioside biosynthesis, sialyltransferase II (ST-II) and N-acetylgalactosaminyltransferase I (GalNAcT), were analyzed upon transient expression of the two enzymes in the neuroblastoma cell lines NG108-15 and F-11. The enzymes were endowed with a C-terminal epitope tag peptide (FLAG) for immunostaining and immunoaffinity purification using a FLAG-specific antibody. Mature ST-II-FLAG and GalNAcT-FLAG were expressed as N-glycoproteins with noncomplex oligosaccharides. ST-II-FLAG was distributed to the Golgi apparatus, whereas GalNAcT-FLAG was found in the ER and Golgi. Inhibition of early N-glycoprotein processing with castanospermine resulted in a distribution of ST-II-FLAG to the ER, whereas that of GalNAcT-FLAG remained unaltered. In contrast to GalNAcT, the activity of ST-II and the amount of immunostained enzyme were reduced concomitantly by 75% upon incubation with castanospermine. This was due to a fourfold increased turnover of ST-II-FLAG, which was not found with GalNAcT-FLAG. The ER retention and increased turnover of ST-II-FLAG were most likely due to its inability to bind to calnexin upon inhibition of early N-glycoprotein processing. Calnexin binding was not observed for GalNAcT-FLAG, indicating a differential effect of N-glycosylation on the turnover and subcellular localization of the two glycosyltransferases.

A novel expression vector for production of epitope-tagged recombinant Fab fragments in bacteria

Labeling of recombinant Fab molecules is an important yet cumbersome and time-consuming procedure that is needed in many immunological experimental designs. This work describes the development of a novel expression vector fusing to the carboxyterminal of the Fab heavy chain fragments a tag peptide (FLAG) that is consistently recognized by a mouse monoclonal antibody. The presence of the FLAG peptide does not alter the binding characteristics of the unmodified Fab molecule, as demonstrated by relative affinity determinations and competition experiments. This new method is suitable for extensive utilization in immunological experimental work using recombinant Fabs.