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)
