PEI是基因转染中常用的高分子载体，同时也是基因转染的“金标准”。PEI转染的原理是带正电的阳离子聚合物与核酸中带负电的磷酸基团形成带正电的复合物后，再与细胞表面带负电的蛋白多糖相互作用，通过细胞胞吞进入细胞。PEI主链上每三个原子就含有一个氮原子，因此具有较高的阳离子密度，这对于结合带负电荷的核酸分子是非常有利的。在生理pH条件下，PEI部分胺基被质子化，随着体系pH值的降低，PEI剩余的胺基被质子化，从而赋予了其出色的核酸结合能力和质子缓冲能力，有利于通过质子海绵效应从内涵体逃逸。但是，PEI高的阳离子电荷密度，导致其严重的细胞毒性。同时，PEI 25K含有4%-11％的残留丙酰基，这可能阻碍聚合物主链与DNA的有效结合，影响基因转染效率。因此，针对PEI的研究工作主要聚焦在通过功能化修饰以及脱丙酰基，来提高转染效率、降低细胞毒性。
Branched PEI Transfection Reagent (MW 25000) 是一种基于分子量25 kDa PEI的DNA转染试剂。通过对PEI进行功能改性，NEO-PEI 丙酰基完全脱落，显著改善了PEI的基因递送性能，表现出较好的稳定性和较低的细胞毒性。Branched PEI Transfection Reagent (MW 25000) 与PEI 25000转染试剂相比，具有很多优点。包括：
(1)Branched PEI Transfection Reagent (MW 25000) 为即用型转染试剂，无需配置，可直接用于细胞转染实验，而PEI 25000需要先把水调成弱酸性助其溶解，溶解后再用NaOH把pH调至中性，配置过程繁琐复杂，容易引入误差导致转染效果不稳定。PEI 40000虽然较PEI 25000更易溶解，但也同样存在配置繁琐，配置过程中容易引入误差的风险。
(2)PEI 25000含有4%-11%的丙酰基残留，丙酰基会阻止聚合物主链与DNA结合，影响转染效果。相较于PEI 25000，Branched PEI Transfection Reagent (MW 25000) 丙酰基完全脱落，因此其性能始终高效如一。
Branched PEI Transfection Reagent (MW 25000) 是一种功能强大、值得信赖且经济实惠的瞬时转染试剂，广泛适用于多种细胞系，包括HEK-293、HEK-293T、CHO-K1、COS-1、COS-7、NIH/3T3、Sf9、HepG2和HeLa细胞等。同时，Branched PEI Transfection Reagent (MW 25000) 在大规模重组蛋白表达和病毒生产等领域有着高效、低毒、稳定、可靠等显著优势。Branched PEI Transfection Reagent (MW 25000) 提供从96孔板到200L生物反应器持续的高表达转染体系，实现从新药研发到工业化生产的轻松过渡。与大多数细胞转染方案相比，使用Branched PEI Transfection Reagent (MW 25000) 既能得到稳定的基因表达效率，又可显著降低转染成本。
产品特点：
⁕ 易于使用——即用型转染试剂，无需配置，可直接用于细胞转染实验。
⁕ 转染效率高——针对多种类型细胞，均表现出优秀的转染效率和高重组蛋白表达。
⁕ 细胞毒性低——作用温和，能更好地实现高转染效率与低细胞毒性之间的平衡。
⁕ 性能更稳定——丙酰基完全脱落，性能始终高效如一。
⁕ 高性价比——既能得到稳定的基因表达效率，又可显著降低转染成本。
⁕ 化学成分明确，不含动物来源成分。
适用范围：
Branched PEI Transfection Reagent (MW 25000) 广泛适用于多种细胞系的DNA转染，可应用于大规模重组蛋白表达和病毒生产等领域。本试剂提供从96孔板到200L生物反应器持续的高表达转染体系，实现从新药研发到工业化生产的轻松过度。
实验流程概要：
图1. Branched PEI Transfection Reagent (MW 25000) 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
(1) 贴壁细胞：转染前一天，将胰酶消化后的细胞按照每孔0.5-1.5x10⁵个细胞的量进行铺板，使其在转染时密度为50%左右。
(2) 悬浮细胞：转染当天，配制转染试剂-核酸复合物之前，在24孔板中进行细胞铺板，每500 µL生长培养基中加入1-3x10⁵个细胞。
⁕ 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-核酸复合物
(1) 取1 μg质粒加入到1.5 mL离心管中，往管中加入50 μL无血清基础培养基(与培养体系一致，无血清)，轻轻混匀，标记为“A”。
(2) 取2.5 μL*转染试剂加入到另一个1.5 mL离心管中，往管中加入50 μL无血清基础培养基(与培养体系一致，无血清)，轻轻混匀，标记为“B”。
⁕ 转染试剂使用量受细胞类型及其他实验条件影响，通常情况下，DNA(μg)和转染试剂(μL)的推荐使用比例为1:2.5。建议初次使用时，可在1:1-1:5的比例范围内设置梯度进行预实验，以达到最优转染效果。
(3) 将“A”和“B”轻轻混合均匀(总体积为100 µL)，在室温下静置30分钟，形成转染试剂-核酸复合物。
⁕ 转染试剂-核酸复合物在室温下4个小时内保持稳定。
3.细胞转染
从培养箱中取出细胞，将上述100 µL转染试剂-核酸复合物加入每孔细胞，将细胞放回培养箱中继续培养。
4.分析转染细胞
转染细胞培养36-48小时后，可根据实际情况用荧光检测法、Western Blot、RT-PCR、ELISA、流式细胞术、报告基因等检测转染效果，或加入筛选药物进行稳定细胞株的筛选。
表一：转染量度标准
细胞培养装置规格 | 培养体系 (mL) | DNA (μg) | 转染试剂 (μL) | 无血清基础培养基体积 (μL) |
96孔板 | 0.1 | 0.2 | 0.5 | 10 |
24孔板
| 0.5 | 1 | 2.5 | 50 |
12孔板
| 1 | 2 | 5 | 100 |
6孔板
| 2 | 2-4 | 5-10 | 200 |
60mm 皿 | 4 | 3-5 | 7.5-12.5 | 400 |
100mm 皿 | 10 | 5-10 | 12.5-25 | 1000 |
125mL 摇瓶 | 30-35 | 30-35 | 75-87.5 | 3000 |
500mL 摇瓶 | 120-140 | 120-140 | 300-350 | 12000 |
1000mL 摇瓶 | 240-280 | 240-280 | 600-700 | 24000 |
注意事项：
1.核酸质量：想要获得最高的转染效率和最低的细胞毒性，应选用高纯度、无菌、无污染、无内毒素的优质核酸。质粒中的内毒素是转染的大敌，内毒素会导致转染效率显著下降，特别是对内毒素敏感的细胞，例如原代细胞、悬浮细胞、造血细胞等。推荐使用无内毒素质粒抽提试剂盒进行质粒提取，保证质粒A₂₆₀/A₂₈₀比值为1.8-2.0。同时，需合理计算质粒用量，转染过量的质粒可能会导致细胞毒性甚至死亡。
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小时内、细胞密度约为50%时进行转染。不同的细胞转染实验，对细胞密度的要求不尽相同。在进行不同核酸或不同细胞系的转染时，需要根据说明书优化实验条件。此外，在实验过程中应保证相同的接种条件，确保实验数据的可重复性。
4. 质粒与转染试剂的使用比例：对于大多数细胞系，转染复合物中DNA(μg)与转染试剂(μL)的比例在1:1至1:5之间，推荐比例为1:2.5。想要获得最优的转染结果，需要对这一比例进行优化，根据所转染细胞及质粒选择适合的转染比例。
5. Branched PEI Transfection Reagent (MW 25000) 可用于有血清培养基的转染，转染后不需要去除复合物或换培养基。但是，制备转染复合物时要求用无血清基础培养基稀释DNA和转染试剂，因为血清会影响复合物的形成。
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该产品的相容性。
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，将图2所示3种状态良好的细胞接种于24孔板，使其在转染时密度约为50%。
2.按照每孔计量，取1μg GFP质粒加入到1.5 mL离心管中，加入50 μL无血清基础DMEM培养基稀释质粒，标记为A。取2.5 μL转染试剂(B1505994)(图2上)或某P公司品牌PEI(图2下)加入到另一个1.5 mL离心管中，加入50 μL无血清基础DMEM培养基稀释转染试剂，标记为B。将A和B轻轻混合均匀，室温静置30分钟，形成转染试剂-核酸复合物。
3.30分钟后，从培养箱中取出细胞，往细胞中加入上述100 µL 转染试剂-核酸复合物，将细胞放回培养箱中继续培养。
4.转染细胞培养48小时后，用荧光显微镜检测GFP绿色荧光蛋白表达情况，并拍照记录，实验结果如图2所示。
图2. 采用Branched PEI Transfection Reagent (MW 25000) 及某P公司品牌PEI转染GFP质粒，转染48小时后，采用荧光显微镜检测GFP绿色荧光蛋白表达情况， B1505994表现出更优秀的转染效率。
PEI (Polyethyleneimine) is a commonly used polymeric vector in gene transfection and also serves as the "gold standard" for this technique. The principle of PEI-mediated transfection is as follows: positively charged cationic polymers form positively charged complexes with the negatively charged phosphate groups in nucleic acids; these complexes then interact with the negatively charged proteoglycans on the cell surface and enter the cell through endocytosis. Every three atoms in the PEI backbone contain one nitrogen atom, giving it a high cationic density—this is highly favorable for binding negatively charged nucleic acid molecules. Under physiological pH conditions, some of the amine groups in PEI are protonated; as the pH of the system decreases, the remaining amine groups in PEI become protonated. This endows PEI with excellent nucleic acid-binding ability and proton buffering capacity, facilitating its escape from endosomes via the proton sponge effect. However, the high cationic charge density of PEI leads to significant cytotoxicity. Additionally, PEI 25K (PEI with a molecular weight of 25,000 Da) contains 4%–11% residual propionyl groups, which may hinder the effective binding between the polymer backbone and DNA, thereby affecting gene transfection efficiency. Therefore, research on PEI primarily focuses on improving transfection efficiency and reducing cytotoxicity through functional modification and depropionylation.
Branched PEI Transfection Reagent (MW 25000) is a DNA transfection reagent based on PEI with a molecular weight of 25 kDa. Through functional modification of PEI, the propionyl groups in NEO-PEI are completely removed, which significantly improves the gene delivery performance of PEI, exhibiting better stability and lower cytotoxicity. Compared with PEI 25000 transfection reagent, Branched PEI Transfection Reagent (MW 25000) has many advantages, including:
(1) Branched PEI Transfection Reagent (MW 25000) is a ready-to-use transfection reagent. It requires no preparation and can be directly used in cell transfection experiments. In contrast, PEI 25000 needs to be dissolved in weakly acidic water first to aid dissolution, and then the pH is adjusted to neutral with NaOH. The preparation process is cumbersome and complicated, and it is easy to introduce errors, resulting in unstable transfection effects. Although PEI 40000 is more soluble than PEI 25000, it also has the problems of cumbersome preparation and the risk of introducing errors during the preparation process.
(2) PEI 25000 contains 4%-11% residual propionyl groups, which will prevent the polymer backbone from binding to DNA and affect the transfection effect. Compared with PEI 25000, the propionyl groups in Branched PEI Transfection Reagent (MW 25000) are completely removed, so its performance is always efficient and consistent.
Branched PEI Transfection Reagent (MW 25000) is a powerful, reliable, and cost-effective transient transfection reagent. It is widely applicable to a variety of cell lines, including HEK-293, HEK-293T, CHO-K1, COS-1, COS-7, NIH/3T3, Sf9, HepG2, and HeLa cells. Meanwhile, Branched PEI Transfection Reagent (MW 25000) exhibits significant advantages such as high efficiency, low toxicity, stability, and reliability in fields like large-scale recombinant protein expression and virus production. It provides a consistent high-expression transfection system ranging from 96-well plates to 200L bioreactors, enabling a seamless transition from new drug research and development to industrial-scale production. Compared with most cell transfection protocols, using Branched PEI Transfection Reagent (MW 25000) not only achieves stable gene expression efficiency but also significantly reduces transfection costs.
Product Features:
⁕ Easy to use – Ready-to-use transfection reagent that requires no preparation and can be directly used in cell transfection experiments.
⁕ High transfection efficiency – Demonstrates excellent transfection efficiency and high recombinant protein expression across multiple cell types.
⁕ Low cytotoxicity – Exhibits a mild effect, enabling a better balance between high transfection efficiency and low cytotoxicity.
⁕ More stable performance – Complete removal of propionyl groups ensures consistently efficient performance.
⁕ High cost-effectiveness – Achieves stable gene expression efficiency while significantly reducing transfection costs.
⁕ Well-defined chemical composition, free of animal-derived components.
Scope of Application:
Branched PEI Transfection Reagent (MW 25000) is widely applicable for DNA transfection in a variety of cell lines and can be used in fields such as large-scale recombinant protein expression and virus production. This reagent provides a consistent high-expression transfection system ranging from 96-well plates to 200L bioreactors, enabling a seamless transition from new drug research and development to industrial-scale production.
Summary of Experimental Procedure:
Figure 1. Experimental Flowchart of Transfection with Branched PEI Transfection Reagent (MW 25000)
Experimental Steps: (Taking a 24-well plate as an example; refer to Table 1: Transfection Volume Standards for sample volumes of other culture plates)
1. Prepare cells for transfection
(1) Adherent cells
One day before transfection, seed the trypsin-digested cells at a density of 0.5–1.5×10⁵ cells per well, so that the cell confluency reaches approximately 50% at the time of transfection.
(2) Suspension cells
On the day of transfection, before preparing the transfection reagent-nucleic acid complex, seed the cells in a 24-well plate. Add 1–3×10⁵ cells to 500 µL of growth medium per well.
⁕ 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 survival rate > 90% for transfection.
2. Prepare the transfection reagent-nucleic acid complex
(1) Add 1 μg of plasmid to a 1.5 mL centrifuge tube, then add 50 μL of serum-free basal medium (consistent with the culture system, serum-free) to the tube. Mix gently and label this tube as "A".
(2) Add 2.5 μL* of transfection reagent to another 1.5 mL centrifuge tube, then add 50 μL of serum-free basal medium (consistent with the culture system, serum-free) to the tube. Mix gently and label this tube as "B".
⁕ The amount of transfection reagent used is affected by cell type and other experimental conditions. Generally, the recommended ratio of DNA (μg) to transfection reagent (μL) is 1:2.5. For first-time use, it is recommended to set up gradients within the ratio range of 1:1 to 1:5 for pre-experiments to achieve the optimal transfection effect.
(3) Gently mix "A" and "B" uniformly (total volume: 100 µL), and let stand at room temperature for 30 minutes to form the transfection reagent-nucleic acid complex.
⁕ The transfection reagent-nucleic acid complex remains stable at room temperature for up to 4 hours.
3. Cell transfection
Take the cells out of the incubator, add 100 µL of the aforementioned transfection reagent-nucleic acid complex to each well of cells, and place the cells back into the incubator for continued culture.
4. Analyze transfected cells
After culturing the transfected cells for 36–48 hours, detect the transfection effect using methods such as fluorescence detection, Western Blot, RT-PCR, ELISA, flow cytometry, or reporter gene assays according to actual conditions. Alternatively, add selection drugs to screen for stable cell lines.
Table 1: Transfection Volume Standards
Specifications of Cell Culture Equipment | Culture System (mL) | DNA (μg) | Transfection Reagent (μL) | Volume of Serum-Free Basal Medium (μL) |
96-well plate | 0.1 | 0.2 | 0.5 | 10 |
24-well plate
| 0.5 | 1 | 2.5 | 50 |
12-well plate
| 1 | 2 | 5 | 100 |
6-well plate
| 2 | 2-4 | 5-10 | 200 |
60mm Dish | 4 | 3-5 | 7.5-12.5 | 400 |
100mm Dish | 10 | 5-10 | 12.5-25 | 1000 |
125mL Shake Flask | 30-35 | 30-35 | 75-87.5 | 3000 |
500mL Shake Flask | 120-140 | 120-140 | 300-350 | 12000 |
1000mL Shake Flask | 240-280 | 240-280 | 600-700 | 24000 |
Precautions:
1. Nucleic acid quality: To achieve the highest transfection efficiency and lowest cytotoxicity, high-quality nucleic acids that are highly pure, sterile, contamination-free, and endotoxin-free should be used. Endotoxin in plasmids is a major obstacle to transfection, as it can significantly reduce transfection efficiency—this is particularly true for endotoxin-sensitive cells, such as primary cells, suspension cells, and hematopoietic cells. It is recommended to use an endotoxin-free plasmid extraction kit for plasmid isolation to ensure the plasmid has an A₂₆₀/A₂₈₀ ratio of 1.8–2.0. Meanwhile, the amount of plasmid used should be calculated appropriately; excessive plasmid for transfection may cause cytotoxicity or even cell death.
2. Cell quality: Cell status greatly affects transfection efficiency. It is recommended to use cells that are in the exponential growth phase with a survival rate of > 90% for transfection.
3. Cell density: Transfection is recommended to be performed within 12–24 hours after cell passage when the cell density is approximately 50%. Different cell transfection experiments have different requirements for cell density. When transfecting different nucleic acids or different cell lines, experimental conditions need to be optimized according to the instructions. In addition, consistent seeding conditions should be maintained during the experiment to ensure the reproducibility of experimental data.
4. Ratio of plasmid to transfection reagent: For most cell lines, the ratio of DNA (μg) to transfection reagent (μL) in the transfection complex ranges from 1:1 to 1:5, with a recommended ratio of 1:2.5. To obtain optimal transfection results, this ratio needs to be optimized, and a suitable transfection ratio should be selected based on the transfected cells and plasmids.
5. Branched PEI Transfection Reagent (MW 25000) can be used for transfection in serum-containing medium, and there is no need to remove the complex or change the medium after transfection. However, when preparing the transfection complex, it is required to dilute DNA and the transfection reagent with serum-free basal medium, as serum can affect the formation of the complex.
6. Since certain components in some special media may inhibit cationic polymer-mediated transfection, it is necessary to test the compatibility of special media with this product.
7. For your health and safety, please operate in accordance with standard procedures and wear a lab coat and gloves when conducting experiments.
8. 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 the 3 types of cells in good condition (as shown in Figure 2) into a 24-well plate, so that their density reaches approximately 50% at the time of transfection.
2. For each well: Add 1 μg of GFP plasmid to a 1.5 mL centrifuge tube, then add 50 μL of serum-free basal DMEM medium to dilute the plasmid, and label this tube as "A". Add 2.5 μL of transfection reagent (Cat. No. B1505994) (top of Figure 2) or PEI from a certain brand P (bottom of Figure 2) to another 1.5 mL centrifuge tube, then add 50 μL of serum-free basal DMEM medium to dilute the transfection reagent, and label this tube as "B". Gently mix "A" and "B" uniformly, and let the mixture stand at room temperature for 30 minutes to form the transfection reagent-nucleic acid complex.
3. After 30 minutes, take the cells out of the incubator, add 100 μL of the aforementioned transfection reagent-nucleic acid complex to the cells, and place the cells back into the incubator for continued culture.
4. After culturing the transfected cells for 48 hours, use a fluorescence microscope to detect the expression of GFP (green fluorescent protein), take photos for documentation, and the experimental results are shown in Figure 2.
Figure 2. GFP plasmid transfection using Branched PEI Transfection Reagent (MW 25000) and PEI from a certain brand P. After 48 hours of transfection, the expression of GFP (green fluorescent protein) was detected by fluorescence microscopy, and B1505994 showed superior transfection efficiency.
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