TPNs were formulated as above at a 15:15:1 peptide:PEG:siRNA ratio at a final DyLight 677-siLuc concentration of 200 nM, and poly-glutamic acid (Sigma; 3,000–15,000 molecular weight) was added post-formulation for a final molar ratio of 0–480:1 polyglutamic acid:siRNA. The particles were incubated for 15 minutes at room temperature prior to gel mobility shift assay, performed as above.
Poly glutamic acid
Manufactured by Merck Group
Poly-glutamic acid is a naturally occurring biopolymer composed of glutamic acid monomers. It serves as a versatile laboratory reagent that can be used in various applications, including cell culture, protein purification, and drug delivery systems.
Automatically generated - may contain errors
5 protocols using poly glutamic acid
1
Optimized siRNA Delivery Formulation
2
Quantitative Nucleosome-Condensin Binding Assay
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200 nM Alexa647 labeled 196 bp mononucleosomes were mixed with 0.0008% poly-glutamic acid (Sigma; Stein and Künzler, 1983 (link)) and half was mixed with 400 nM recombinant xH1.8 in 1× binding buffer (10 mM HEPES pH 8, 50 mM NaCl, 2.5 mM MgCl2, 5 mM ATP, 0.5 mM DTT, 0.05% Triton X-100) and incubated for 30 min at room temperature. 100 nM of the mononucleosomes with or H1.8 were mixed with the indicated concentration of condensin I in 1× binding buffer at 4°C for 30 min and subject to electrophoresis onto a 5% polyacrylamide gel in 0.5× TBE at room temperature. The gels were imaged on a LI-COR Odyssey (LI-COR Biotechnology). The binding curves were fitted using GraphPad Prism 8.4.3 using the sigmoidal binding curve option of the nonlinear curve fitting.
3
Polymer Coatings for Electron Microscopy
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The following polymers were tested as grid coatings: polyglutamic acid with an average molecular weight of 50 000–100 000 Da (Sigma–Aldrich), poly(sodium 4-styrenesulfonate) with an average molecular weight of 200 000 Da (Sigma–Aldrich), polyethylene glycol with an average molecular weight of 35 000 Da (Sigma–Aldrich) and poly(l -lysine) hydrobromide (Sigma–Aldrich) with an average molecular weight of 70 000–150 000 Da. The polyelectrolytes were diluted to a final concentration of 10 mg ml−1 in nuclease-free water and were used in the preparation of polymer-coated grids with the same protocol as that used for the ssDNA-coated grids.
4
Peptide Synthesis and Purification
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C17 and AK16 peptides were synthesized on 9050 peptide synthesizers (PerSeptive). Fluorenylmethyloxy-carbonyl (Fmoc) amino acids were coupled onto the PAL resin (PerSeptive) so that the resultant peptides have an amide group at the C terminus7 (link). For coupling the Cys residue, N-Fmoc-S-acetamidomethylcysteine was used, leaving the S-acetamidomethyl group in the peptides. The peptides were purified by reverse-phase HPLC (Waters μBondasphere C18-100 Å 19×150 mm)7 (link).
Polyglutamic acid was purchased from Sigma (Product No. P-1818, Mol. Weight; 1,500–3,000), and was used without further purification.
GuHCl was of ultrapure reagent grade from ICN Biomedicals, Inc. (Lot 2345B). Its concentration was calibrated by refractive index measurements. All other reagents were of guaranteed reagent grade for this study.
Temperature was controlled by a ULT-80 controller (NESLAB). Viscosities were measured by a syringe viscometer and calibrated by standard solution (JS 5 and JS 20 of Nihon Grease Co.).
Polyglutamic acid was purchased from Sigma (Product No. P-1818, Mol. Weight; 1,500–3,000), and was used without further purification.
GuHCl was of ultrapure reagent grade from ICN Biomedicals, Inc. (Lot 2345B). Its concentration was calibrated by refractive index measurements. All other reagents were of guaranteed reagent grade for this study.
Temperature was controlled by a ULT-80 controller (NESLAB). Viscosities were measured by a syringe viscometer and calibrated by standard solution (JS 5 and JS 20 of Nihon Grease Co.).
5
VEGF-NP Immobilization on Glass Slides
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Glass slides were treated with Poly-glutamic acid (Sigma-Aldrich) (500 µm/mL) for 15 minutes, rinsed with PBS for 15 minutes followed by addition with VEGF-NPs (3 mg/mL) for 15 minutes at room temperature. Finally, samples were rinsed in PBS for 15 minutes and then with poly-lysine fluorescein-isothiocyanate (PLL-FITC) for 15 minutes. Glass slides were then observed under an epifluorescent microscope (LEICA DM 4000 B).
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