蛋白质转染是将生物学上有活性的蛋白质或肽段导入活细胞，通过替换体内具有缺陷或缺失的蛋白而发挥治疗作用的新型手段。与传统小分子化合物药物相比，蛋白质药物具有更强的特异性和效用，在低浓度时就可以表现出高特异性和高活性。此外，在活细胞内递送蛋白质能够获得比基因转染更迅速的蛋白表达，是核酸转染的替代方式，可作为一种功能强大的研究方法或治疗策略，广泛应用于生命科学领域。
基于肽转导结构域（PTD）的技术已成功开发用于蛋白质的胞内转染。然而，这些PTD与蛋白质的相互作用较差，通常需要蛋白质和PTD之间形成共价连接。目前已开发出多种试剂用于蛋白质的胞内转染，如基于多肽、阳离子脂质和聚合物的转染试剂。这些转染试剂主要是通过非共价相互作用与蛋白质形成复合物，然后通过胞吞作用进入细胞，这些蛋白质复合物通常会进入内涵体中，如果不能及时从内涵体中释放，蛋白质就会在内涵体发展成为溶酶体后，在溶酶体中发生降解。因此，转染试剂应该有很强的破膜活性，从而保证转染蛋白能够从内涵体中释放进入细胞质并发挥作用。
Protein Transfection Reagent是一种基于阳离子高分子聚合物的新型蛋白转染试剂，适用于几百Da的小分子多肽到几百kDa的大分子蛋白质的转染。其原理为带正电的阳离子高分子转染试剂与蛋白质通过静电相互作用、疏水相互作用以及氢键相互作用等多种非共价相互作用的协同作用与蛋白质结合形成带正电的复合物，之后与细胞表面带负电的蛋白多糖相互作用，并通过内吞作用进入细胞。在内涵体熟化过程中，随着pH值的降低，阳离子高分子转染试剂与内涵体膜的相互作用增加，导致内涵体膜的破裂，从而将蛋白质释放入细胞质中发挥作用。由于转染试剂通过非共价形式与蛋白质结合形成复合物，因此不会干扰蛋白质的生物学活性。
Protein Transfection Reagent对多种常见细胞具有高水平转染效率，具有高效低毒、操作简单、重复性好等优点。另外，由于该阳离子高分子转染试剂与蛋白质是通过多种非共价相互作用协同结合蛋白质的，因此可以适用于各种理化性质的蛋白质转染。Protein Transfection Reagent可用于细胞信号传导和凋亡检测、蛋白质-蛋白质相互作用、蛋白质定位和隔室穿梭等各种功能研究。
产品特点：
⁕ 卓越的蛋白转染效率——针对广泛类型的细胞，均表现出卓越的蛋白质转染效率。
⁕ 蛋白活性高——与蛋白质非共价结合，递送后的蛋白和肽仍具有高生物学活性。
⁕ 细胞毒性低——作用温和，生物相容性好。
⁕ 操作简单——简单快速的实验方案，无需分离、克隆和转染基因序列，孵育后3-12小时内可获得稳定的蛋白转染效果。
⁕ 适用范围广——适用于各种理化性质的蛋白质转染。
适用范围：
Protein Transfection Reagent是一种基于阳离子高分子聚合物的新型蛋白转染试剂，对多种常见细胞具有高水平转染效率，适用于几百Da的小分子多肽到几百kDa的大分子蛋白质及各种理化性质的蛋白质转染。
实验流程概要：
图1. Protein Transfection Reagent (P1505996) 转染实验流程图
实验步骤：
(以24孔板为例，其他培养板加样体积参考表一：转染量度标准)
1. 准备待转染细胞
转染前一天，将胰酶消化后的细胞按照每孔1-3x10⁵个细胞的量进行铺板，使其在转染时密度为50%-100%。
⁕ 细胞状态会极大影响转染效率，待转染细胞应处于良好生长状态，建议使用生长处于指数期、存活率大于90% 的细胞进行转染。
2. 准备转染试剂-蛋白质复合物
(1)取1-10 μg 蛋白质加入到1.5 mL离心管中，加入1-3 μL转染试剂与蛋白质混合，室温孵育3分钟。
⁕ 蛋白质的实际使用量需根据蛋白质的类型和具体实验进行优化，不同性质的蛋白质使用量相差较大，例如：荧光蛋白EGFP建议24孔板使用量为5-10 μg，荧光蛋白Phycoerythrin建议24孔板使用量为0.5-2 μg，凋亡蛋白Saporin建议24孔板使用量为0.1-1 μg。
⁕ 转染试剂的用量应根据目的细胞类型、接种细胞密度、培养皿的大小进行调整，请参见表一。
(2)往上述混合物中加入100 μL无血清基础培养基1(与培养体系一致，无血清)，轻轻混匀，在室温下静置20分钟，形成转染试剂-蛋白质复合物。
(3)20分钟后，往转染试剂-蛋白质复合物中加入400 μL无血清基础培养基2(与培养体系一致，无血清)稀释复合物。
3.蛋白质转染
弃去细胞培养基，用预热的PBS清洗细胞1-2次，将上述500 µL转染试剂-蛋白复合物加入每孔细胞进行蛋白质转染。
4.分析蛋白质转染效果
转染试剂-蛋白复合物与细胞孵育3-12小时后，可根据实际情况，用荧光检测法、流式细胞术、Western Blot、免疫荧光染色等实验方法进行后续检测及分析。
表一：转染量度标准
细胞培养装置规格 | 培养体系 (mL) | 转染试剂 (μL) | 无血清基础培养基1 (μL) | 无血清基础培养基2 (μL) |
96孔板
| 0.1 | 0.2-0.6
| 20
| 80 |
24孔板
| 0.5 | 1-3
| 100
| 400 |
12孔板
| 1 | 2-6
| 200
| 800 |
6孔板
| 2 | 4-12
| 400
| 1600 |
60mm 皿
| 4 | 8-24
| 800
| 3200 |
100mm 皿
| 10 | 20-60
| 2000
| 8000 |
注意事项：
1.蛋白质质量：蛋白质中存在的杂质、污染物和添加剂都可能会影响蛋白质递送效率，我们建议使用尽可能高纯度的蛋白质样品。
2.细胞质量：细胞状态会极大影响转染效率，建议使用生长处于指数期、存活率大于90%的细胞进行转染。
3. 细胞密度：建议细胞传代后12-24 h内、细胞密度为50%-100% 时进行转染。不同的蛋白转染实验，对细胞密度的要求不尽相同。在进行不同蛋白质或不同细胞系的转染时，需要根据说明书再次优化实验条件。此外，在实验过程中需保证相同的接种条件，确保实验数据的可重复性。
4. 蛋白质与转染试剂使用量：蛋白质的实际使用量需根据蛋白质的类型和具体实验进行优化，不同性质的蛋白质使用量相差较大。转染试剂的用量应根据目的细胞类型、接种细胞密度、培养皿的大小进行调整(参见表一)。想要获得最优的蛋白递送效果，需对蛋白质及转染试剂的使用量进行优化，选择适合的剂量。
5. 采用无血清转染体系进行蛋白质转染可获得高效的蛋白质递送效果，这是因为血清中的血清蛋白会通过竞争结合蛋白转染试剂，从而影响转染试剂-蛋白质复合物的形成和稳定性。
6. 由于一些特殊培养基中的某些成分可能会抑制阳离子聚合物介导的转染，因此有必要检测特殊培养基与本产品的相容性。
7. 为了您的健康安全，请规范操作，穿戴实验服与手套开展实验。
8. 本产品仅供科研使用，请勿用于临床诊断及治疗。
实验案例分析：
1.转染前一天，将CHO、NIH-3T3、H1299和HEK293细胞分别接种于24孔板，使其在转染时密度约为95%。
2.按照每孔计量，取10 μg EGFP(1mg/mL)蛋白加入到1.5 mL离心管中，再分别加入1 μL、2 μL、3 μL 转染试剂(P1505996)与蛋白质混合，室温孵育3 分钟。
3.往混合物中加入100 μL无血清基础DMEM培养基，轻轻混匀，在室温下静置20 分钟，形成转染试剂-蛋白质复合物。
4.20分钟后，往复合物中加入400 μL无血清基础DMEM培养基稀释复合物。
5.弃去细胞培养基，用预热的PBS清洗细胞2次，将上述500 µL转染试剂-蛋白质复合物加入每孔细胞，与细胞孵育4小时。
6.转染4小时后，用荧光显微镜检测EGFP绿色荧光蛋白转染效果，实验结果如图2所示。
图2. 采用Protein Transfection Reagent (P1505996)转染绿色荧光蛋白EGFP(10 μg)，4小时后用荧光显微镜检测EGFP的表达情况。
Protein transfection is a novel approach that involves delivering biologically active proteins or peptide fragments into living cells. It exerts therapeutic effects by replacing defective or missing proteins in the body. Compared with traditional small-molecule compound drugs, protein drugs exhibit greater specificity and efficacy, and can demonstrate high specificity and high activity even at low concentrations. Additionally, delivering proteins into living cells enables faster protein expression than gene transfection. As an alternative to nucleic acid transfection, it serves as a powerful research method or therapeutic strategy and is widely applied in the field of life sciences.
Technologies based on Peptide Transduction Domains (PTDs) have been successfully developed for the intracellular transfection of proteins. However, these PTDs exhibit poor interaction with proteins and typically require the formation of covalent bonds between the protein and the PTD. Currently, a variety of reagents have been developed for intracellular protein transfection, such as transfection reagents based on polypeptides, cationic lipids, and polymers. These transfection reagents primarily form complexes with proteins through non-covalent interactions, and then enter cells via endocytosis. These protein complexes usually traffic to endosomes; if they cannot be released from endosomes in a timely manner, the proteins will be degraded in lysosomes after the endosomes mature into lysosomes. Therefore, transfection reagents should possess strong membrane-disrupting activity to ensure that the transfected proteins can be released from endosomes into the cytoplasm and exert their functions.
Protein Transfection Reagent is a novel protein transfection reagent based on cationic macromolecular polymers. It is suitable for transfecting small-molecule peptides (hundreds of Da in molecular weight) to large-molecule proteins (hundreds of kDa in molecular weight). Its mechanism is as follows: the positively charged cationic macromolecular transfection reagent binds to proteins through the synergistic effect of multiple non-covalent interactions—including electrostatic interactions, hydrophobic interactions, and hydrogen bond interactions—to form positively charged complexes. Subsequently, these complexes interact with the negatively charged proteoglycans on the cell surface and enter the cell via endocytosis. During endosome maturation, as the pH value decreases, the interaction between the cationic macromolecular transfection reagent and the endosomal membrane increases, leading to the rupture of the endosomal membrane. This allows the protein to be released into the cytoplasm to exert its biological function. Since the transfection reagent binds to the protein in a non-covalent manner to form complexes, it does not interfere with the biological activity of the protein.
Protein Transfection Reagent exhibits high transfection efficiency in a variety of common cell types, with advantages including high efficiency, low toxicity, simple operation, and good reproducibility. Additionally, since this cationic macromolecular transfection reagent binds to proteins through the synergistic effect of multiple non-covalent interactions, it is suitable for transfecting proteins with diverse physicochemical properties. Protein Transfection Reagent can be used in various functional studies, such as cell signal transduction and apoptosis detection, protein-protein interaction analysis, protein localization, and compartmental shuttling research.
Product Features:
⁕ Excellent protein transfection efficiency — Demonstrates outstanding protein transfection efficiency across a wide range of cell types.
⁕ High protein activity — Binds to proteins via non-covalent interactions, ensuring that the delivered proteins and peptides retain high biological activity.
⁕ Low cytotoxicity — Exhibits gentle action and good biocompatibility.
⁕ Simple operation — Features a simple and rapid experimental protocol; no need for isolation, cloning, or transfection of gene sequences. Stable protein transfection results can be achieved within 3–12 hours after incubation.
⁕ Broad applicability — Suitable for transfecting proteins with diverse physicochemical properties.
Scope of Application:
Protein Transfection Reagent is a novel protein transfection reagent based on cationic macromolecular polymers. It exhibits high transfection efficiency in a variety of common cell types and is suitable for transfecting small-molecule peptides (hundreds of Da in molecular weight) to large-molecule proteins (hundreds of kDa in molecular weight), as well as proteins with diverse physicochemical properties.
Summary of Experimental Procedure:
Figure 1. Experimental Flowchart of Protein Transfection Reagent (P1505996)-Mediated Transfection
Experimental Procedures:
(Using a 24-well plate as an example; refer to Table 1: Transfection Volume Standards for sample volumes in other culture plates)
1. Prepare Cells for Transfection
One day before transfection, seed the trypsin-digested cells at a density of 1–3×10⁵ cells per well, so that the cell confluency reaches 50%–100% at the time of transfection.
⁕ Cell status significantly affects transfection efficiency. Cells to be transfected should be in a good growth state. It is recommended to use cells that are in the exponential growth phase with a viability of >90% for transfection.
2. Prepare Transfection Reagent-Protein Complexes
(1) Add 1–10 μg of protein to a 1.5 mL centrifuge tube, then add 1–3 μL of transfection reagent, mix with the protein, and incubate at room temperature for 3 minutes.
⁕ The actual amount of protein used needs to be optimized based on the protein type and specific experiment, as the required amount varies significantly among proteins with different properties. For example:
For the fluorescent protein EGFP, the recommended amount for a 24-well plate is 5–10 μg;
For the fluorescent protein Phycoerythrin, the recommended amount for a 24-well plate is 0.5–2 μg;
For the apoptotic protein Saporin, the recommended amount for a 24-well plate is 0.1–1 μg.
⁕ The amount of transfection reagent should be adjusted according to the target cell type, seeded cell density, and the size of the culture dish. Please refer to Table 1.
(2) Add 100 μL of serum-free basal medium 1 (consistent with the culture system, serum-free) to the above mixture, mix gently, and stand at room temperature for 20 minutes to form the transfection reagent-protein complex.
(3) After 20 minutes, add 400 μL of serum-free basal medium 2 (consistent with the culture system, serum-free) to the transfection reagent-protein complex to dilute the complex.
3. Protein Transfection
Aspirate the cell culture medium, wash the cells 1–2 times with pre-warmed PBS, and add 500 µL of the aforementioned transfection reagent-protein complex to each well for protein transfection.
4. Analysis of Protein Transfection Efficacy
After incubating the transfection reagent-protein complex with the cells for 3–12 hours, subsequent detection and analysis can be performed using experimental methods such as fluorescence detection, flow cytometry, Western Blot, and immunofluorescence staining, depending on the actual experimental conditions.
Table 1: Transfection Volume Standards
Specifications of Cell Culture Equipment | Culture System (mL) | Transfection Reagent (μL) | Serum-Free Basal Medium 1 (μL) | Serum-Free Basal Medium 2 (μL) |
96-well plate
| 0.1 | 0.2-0.6
| 20
| 80 |
24-well plate
| 0.5 | 1-3
| 100
| 400 |
12-well plate
| 1 | 2-6
| 200
| 800 |
6-well plate
| 2 | 4-12
| 400
| 1600 |
60mm Dish
| 4 | 8-24
| 800
| 3200 |
100mm Dish
| 10 | 20-60
| 2000
| 8000 |
Precautions:
1. Protein Quality: Impurities, contaminants, and additives present in proteins may affect protein delivery efficiency. We recommend using protein samples with the highest possible purity.
2. Cell Quality: Cell status significantly impacts transfection efficiency. It is advisable to use cells that are in the exponential growth phase with a viability of >90% for transfection.
3. Cell Density: Transfection is recommended to be performed within 12–24 hours after cell passage, when the cell density reaches 50%–100%. Different protein transfection experiments have varying requirements for cell density. When transfecting different proteins or cell lines, experimental conditions need to be re-optimized according to the instruction manual. Additionally, consistent seeding conditions must be maintained during the experiment to ensure the reproducibility of experimental data.
4. Dosages of Protein and Transfection Reagent: The actual dosage of protein needs to be optimized based on the protein type and specific experiment, as the required dosage varies greatly among proteins with different properties. The dosage of the transfection reagent should be adjusted according to the target cell type, seeded cell density, and the size of the culture dish (see Table 1). To achieve optimal protein delivery results, it is necessary to optimize the dosages of the protein and transfection reagent and select the appropriate amounts.
5. Serum-Free Transfection System: Using a serum-free transfection system for protein transfection can achieve high-efficiency protein delivery. This is because serum proteins in the serum will competitively bind to the protein transfection reagent, thereby affecting the formation and stability of the transfection reagent-protein complex.
6. Compatibility with Special Media: Since certain components in some special media may inhibit cationic polymer-mediated transfection, it is necessary to test the compatibility between the special medium and this product.
7. Health and Safety: For your health and safety, please operate in a standardized manner and wear a lab coat and gloves when conducting experiments.
8. Intended Use: This product is for research use only and shall not be used for clinical diagnosis or treatment.
Experimental Case Analysis:
1. One day before transfection, seed CHO, NIH-3T3, H1299, and HEK293 cells separately into 24-well plates, ensuring that the cell confluency reaches approximately 95% at the time of transfection.
2. For each well, add 10 μg of EGFP protein (1 mg/mL) to a 1.5 mL centrifuge tube, then add 1 μL, 2 μL, and 3 μL of Transfection Reagent (P1505996) respectively, mix with the protein, and incubate at room temperature for 3 minutes.
3. Add 100 μL of serum-free basal DMEM medium to the mixture, mix gently, and stand at room temperature for 20 minutes to form the transfection reagent-protein complex.
4. After 20 minutes, add 400 μL of serum-free basal DMEM medium to the complex to dilute it.
5. Aspirate the cell culture medium, wash the cells twice with pre-warmed PBS, add 500 μL of the aforementioned transfection reagent-protein complex to each well of cells, and incubate with the cells for 4 hours.
6. Four hours after transfection, use a fluorescence microscope to detect the transfection efficacy of the EGFP green fluorescent protein. The experimental results are shown in Figure 2.
Figure 2. Detection of EGFP Expression via Fluorescence Microscopy 4 Hours After Transfecting Green Fluorescent Protein EGFP (10 μg) Using Protein Transfection Reagent (P1505996)
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