Cell penetrating peptide-mediated peptide nucleic acid oligomer delivery assay
Cell penetrating peptide-mediated peptide nucleic acid oligomer delivery assay
Site-specific knockout and non-toxic DNA delivery technologies are particularly important in the study of protein function and as a means for future therapies. The problem with many of these techniques is that they are biocompatible and toxic, especially when applied in vivo. Certain cationic peptides can displace across cell membranes and, more importantly, can carry "cargo" several times larger than themselves, which can be used to enhance the biocompatibility of hydrophilic macromolecules. These cationic peptides (also commonly known as cell-penetrating peptides) can be used to transport a wide variety of "cargoes", including gene regulatory oligonucleotides and their analogs, both in vitro and in vivo. Author: T. Friedman et al, Translator: Jingwei et al, This experiment is from "Gene Transfer".
Operation method
Peptide binding to PNA (delivery of the binding product to human cells) Move Peptide binding to PNA (delivery of the binding product to human cells) Materials reagents Methods Peptide and PNA are linked by disulfide bonds 1. Weigh 0.5 to 2 mg of peptide and Imol PNA into separate centrifuge tubes. 2- Dissolve the PNA with 200 ul of deoxygenated DMSO, if it does not dissolve, incubate at 55°C for 5 min, if it does not dissolve, use the suspension to continue the experiment. 3- Dissolve the peptide in IOOm 10. Olm 0 l/L acetate buffer (pH 5. 5). 4 . Add 200ul of DMF (recommended) or DMSO to each solution. 5-Mix the two solutions and shake well. If the PNA is still not dissolved, centrifuge for 2 min at 1oooog (maximum speed of a tabletop centrifuge), add 5 to 300ul of TFA (depending on the size of the precipitate), and resuspend after 20s with shaking. If TFA is not available, 6mol/L guanidine hydrochloride or 7mol/L urea can be substituted. Alternatively, PNA solubility can be improved by attaching two lysines to the carboxyl and amino ends. Such disulfide bond formation can be performed in 0.01mol/L acetate buffer and acetonitrile solution (50/50). 6- Shake or stir at room temperature for 2 h or overnight. Avoid direct light. 7-Semi-preparative reversed-phase-HPLC separation of reaction products. a. The gradient depends on the hydrophobicity of the peptide. For hydrophobic proteins, use an isocratic 20 % Wash B for 5 min, followed by 40 min of Wash B with the gradient increased to 100 %; for hydrophobic proteins, use an isocratic 20 % Wash B for 5 min and a gradient of 10 % for 40 min. The HPLC profiles of arginine- and lysine-rich peptides are similar to those of PNA oligomers, making effective separation difficult. An attempt can be made to increase from 0 % Wash B for 60 min to 8 0 % B. b - The detection wavelength is 218 nm and the maximum absorption value of the peptide band is 260 nm, indicating the PN A nucleic acid base. Use 260 nm for single wavelength detection. 8-Collect the peaks that absorb at both wavelengths. 9. Store lyophilized samples in a dark place at 120°C to avoid repeated freezing and thawing. Apply mass spectrometer to analyze the splice to determine the correct product. Deliver the CPP-S^S^P N A conjugate to the cell for antisense application. 10. At least one day prior to the experiment, inoculate the cell line expressing the target protein so that cell confluence reaches 40 % to 60 % on the day of the experiment. Six wells are required (two parallel antisense experiments, two for control PNA, and two controls not manipulated). 11. Change the medium to fresh serum-free medium. 12. Prepare a fresh ○.l m m ○ l/L C P P -S S ^ N A succussion or thaw a block of previously prepared succussion (store at 20°C). Prepare a series of dilutions from O .lmmol/L to 5umol/L to estimate antisense effects at low concentrations. This requires more cells and splice material than listed in this experiment. Heat the C P P -S ^ P N A solution at 13.55°C for lm i n . 14. In cell culture wells, add 10ul of 0.1m m illion l/L C P P -S--S--P N A solution per I m l of medium to a final concentration of lumol/L of C P P -S ^ P N A . 15.37°C for 3 h, then switch to complete medium. 16.37°C for at least 2 times the half-life of the target protein. 17. Collect the cells and determine the amount of protein by appropriate methods. For more product details, please visit Aladdin Scientific website.
