BioVector? pEGFP-C1-PKAgamma Cys1 熒光表達質(zhì)粒 / BioVector? pEGFP-C1-PKAgamma Cys1 Fluorescence Expression Plasmid

通用定義 / General Definition:

BioVector? pEGFP-C1-PKAgamma Cys1 是一種用于在哺乳動物細胞中表達 EGFP（增強型綠色熒光蛋白） 與 PKAgamma（蛋白激酶 A 催化亞基 $\gamma$ 亞型） 融合蛋白的表達載體。在該特定構(gòu)建體中，Cys1 通常指代針對 PKAgamma 序列中第 1 位（或特定位點）半胱氨酸的突變或修飾。pEGFP-C1 骨架提供了強力的 CMV 啟動子，并在融合蛋白的 N 端 帶有 EGFP 標簽。該質(zhì)粒主要用于研究 PKA $\gamma$ 亞型的細胞定位、激酶活性調(diào)控、半胱氨酸介導(dǎo)的氧化還原敏感性以及其在生殖組織（PKA $\gamma$ 主要表達于睪丸）中的特定信號轉(zhuǎn)導(dǎo)功能。

BioVector? pEGFP-C1-PKAgamma Cys1 is a mammalian expression vector designed to produce a fusion protein of EGFP (Enhanced Green Fluorescent Protein) and the $\gamma$ catalytic subunit of Protein Kinase A (PKAgamma). In this specific construct, Cys1 typically refers to a mutation or specific modification of a cysteine residue (potentially at position 1 or a prioritized site) within the PKAgamma sequence. The pEGFP-C1 backbone utilizes a strong CMV promoter and places the EGFP tag at the N-terminus of the fusion. This plasmid is primarily used to investigate the subcellular localization, kinase activity regulation, redox sensitivity involving cysteines, and the tissue-specific signaling roles of the PKA $\gamma$ isoform, which is predominantly expressed in the testes.

BioVector? pEGFP-C1-PKAgamma Cys1 技術(shù)說明書 (Technical Datasheet)

中文版說明書 (Chinese Datasheet)

1. 質(zhì)?；拘畔?/strong>

• 啟動子： CMV (Cytomegalovirus) 啟動子（強力、組成型表達）。

• 融合標簽： EGFP (N-terminal fusion)。

• 插入片段： PKAgamma (PRKACG)，攜帶 Cys1 突變。

• 復(fù)制子： pUC ori。

• 細菌抗性： 卡那霉素 (Kanamycin, 50 $\mu$g/mL)。

• 真核篩選： 新霉素/G418 (Neomycin/G418)。

2. 核心生物學(xué)功能

• 熒光示蹤： EGFP 允許通過熒光顯微鏡實時觀察 PKA $\gamma$ 在活細胞內(nèi)的分布（如細胞核與細胞質(zhì)的穿梭）。

• 半胱氨酸功能研究： Cys1 突變通常用于探索半胱氨酸對蛋白激酶結(jié)構(gòu)的穩(wěn)定性影響或其是否參與形成二硫鍵/氧化還原調(diào)節(jié)。

• 激酶研究： 研究 $\gamma$ 亞型與經(jīng)典 $\alpha$ 或 $\beta$ 亞型在底物特異性上的差異。

3. 實驗應(yīng)用方案

• 細胞轉(zhuǎn)染： 適用于 HEK293T、HeLa 或原代精母細胞等哺乳動物細胞系。

• 免疫共沉淀 (Co-IP)： 利用 EGFP 抗體拉取融合蛋白，分析 PKA $\gamma$ 的相互作用組。

• FRET 實驗： 與表達 PKA 調(diào)節(jié)亞型（R 亞型）的紅色熒光載體共轉(zhuǎn)，研究全酶的解離與激活。

4. 注意事項

• 突變核對： 建議通過 Sanger 測序確認 Cys1 突變的準確性及讀碼框是否對齊。

• 表達強度： 由于 CMV 啟動子極強，過度表達可能導(dǎo)致融合蛋白在胞漿內(nèi)形成非特異性聚集，實驗時可嘗試調(diào)整轉(zhuǎn)染質(zhì)粒量。

English Datasheet

1. Basic Plasmid Information

• Promoter: CMV (Cytomegalovirus) – high-level constitutive expression in most mammalian cells.

• Fusion Tag: EGFP (N-terminal).

• Insert: PKAgamma (PRKACG) with Cys1 mutation/modification.

• Replicon: pUC ori.

• Bacterial Selection: Kanamycin (50 $\mu$g/mL).

• Eukaryotic Selection: Neomycin/G418.

2. Key Biological Functions

• Fluorescent Tracking: The EGFP tag enables real-time visualization of PKA $\gamma$ dynamics and subcellular partitioning (e.g., nucleocytoplasmic shuttling).

• Cysteine Mechanism Studies: The Cys1 variant is utilized to determine the role of specific cysteine residues in kinase folding, structural integrity, or sensitivity to oxidative stress.

• Isoform Specificity: Studying the unique enzymatic properties of the $\gamma$ subunit compared to the ubiquitously expressed $\alpha$ and $\beta$ isoforms.

3. Experimental Applications

• Mammalian Transfection: Highly efficient in common cell lines like HEK293, COS-7, or specialized testicular cell models.

• Protein Interaction Profiling: Using anti-GFP beads for pull-down assays to identify isoform-specific binding partners.

• Live-Cell Imaging: Monitoring kinase redistribution following cAMP stimulation or environmental changes.

4. Key Usage Notes

• Sequence Verification: BioVector? recommends verifying the insert via sequencing using the EGFP-N sequencing primer to ensure the Cys1 mutation and frame are correct.

• Photostability: While EGFP is stable, prolonged imaging at high laser power may cause photobleaching; use anti-fade mounting media for fixed samples or optimized exposure for live imaging.

注意 / Note: 質(zhì)粒應(yīng)儲存在 -20 攝氏度下。反復(fù)凍融可能導(dǎo)致 DNA 降解，建議將高濃度原始質(zhì)粒稀釋至 100 ng/$\mu$L 后分裝備用。

Store the plasmid at -20°C. Repeated freeze-thaw cycles can lead to DNA degradation; BioVector? suggests aliquoting the stock solution into working concentrations (e.g., 100 ng/$\mu$L) to ensure long-term stability.

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