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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