C6 protected siRNA oligomers (IDT technologies) were reduced to their free thiol form using DL-Dithiothreitol (DTT) in 0.1M Triethylammonium bicarbonate (TEAbc) at pH 8.5 and then reacted with dithio-bis-maleimidoethane (DTME) in 300mM NaOAc/acetonitrile at pH 5.2. Purification of the siRNA-DTME intermediate by precipitation with ethanol was followed by reaction with the purified cyclic R11A peptide in 300mM NaOAc/acetonitrile at pH 5.2 with gentle mixing at room temperature. Analytical C-18 RP-HPLC purification of the resulting siRNA-DTME-LTP-Cy5.5 conjugates using triethylamine acetate (TEAA)/Acetonitrile gradients on a Waters Alliance chromatography system was followed by lyophilization and then re-lyophilization from nuclease free water. MALDI-TOF analysis of the purified conjugates on an Applied Biosystems Voyager workstation using 3-hydroxypicolinic acid (3-HPA) matrix in ammonium citrate allowed for confirmation of the expected mass and identity of the final siRNA-cyclic-LTP conjugate.
Voyager workstation
Manufactured by Thermo Fisher Scientific
The Voyager workstation is a lab equipment product designed for sample preparation and processing. It features a compact and modular design to accommodate various laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
5 protocols using voyager workstation
1
Synthesis of Fluorescent siRNA Conjugates
2
Cyclic Cy5.5 Lung Targeting Peptides
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Cyclic Cy5.5 labeled lung targeting peptides (LTPs) were synthesized as above using 2-chlorotrityl resin as solid support. Cleavage of fully-protected peptide fragments were accomplished under mildly acidic conditions followed by head to tail cyclization using 3- (diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) in Dimethylformamide/Dichloromethane (DMF/DCM) for 5 days at room temperature. Final cleavage and deprotection of the cyclized LTP peptides using Trifluoroacetic acid:Triisopropylsilane:H2O (TFA:TIPS:H2O - 90:25:25) was followed by precipitation in Diethyl Ether (EtO2) to isolate the crude peptide. The epsilon amino group of Lysine was then labelled with Cy5.5-NHS in 0.1M Triethylammonium bicarbonate (TEAbc)/acetonitrile at pH 8.5. The resulting Cy5.5 labelled cyclic LTP peptide was then directly purified by preparative C- 18 RP-HPLC on a Waters Delta Prep 4000 chromatography system using standard Acetonitrile/0.1%TFA gradient conditions followed by lyophilization. MALDI-TOF analysis of the purified conjugates on an Applied Biosystems Voyager workstation using α-Cyano-4-hydroxycinnamic acid (CHCA) matrix allowed for confirmation of the expected mass and identity of the final product.
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3
Synthesis and Purification of Cy5.5-Labeled Lung Targeting Peptides
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Linear Cy5.5 labeled lung targeting peptides (LTPs) were synthesized on a Liberty CEM microwave synthesizer using Fluorenylmethyloxycarbonyl (FMOC) chemistry. Stepwise addition of each FMOC protected amino acid was accomplished on an amide resin as solid support using Ethyl-(2Z)-2-cyano-2-hydroxyiminoacetate/N,N-Diisopropylcarbodiimide (Oxyma/DIC) activation chemistry. The N-terminal amino group of the resin bound LTP peptides were then conjugated with Cy5.5-NHS (Lumiprobe Corporation) in DIPEA/DMF. Final cleavage of Cy5.5-LTP from the resin with Trifluoroacetic acid : Triisopropylsilane : H2O (TFA:TIPS:H2O-90:25:25) was followed by precipitation in Diethyl Ether (EtO2). The resulting crude Cy5.5-LTP peptides were purified by semi-preparative C-5 RP-HPLC on a Waters Delta Prep 4000 chromatography system using standard Acetonitrile/0.1%TFA gradient conditions.
MALDI-TOF analysis of the purified conjugates on an Applied Biosystems Voyager workstation using α-Cyano-4-hydroxycinnamic acid (CHCA) matrix allowed for confirmation of the expected mass and identity of the final product.
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MALDI-TOF analysis of the purified conjugates on an Applied Biosystems Voyager workstation using α-Cyano-4-hydroxycinnamic acid (CHCA) matrix allowed for confirmation of the expected mass and identity of the final product.
4
Synthesis and Purification of Cyclic Thiolated Lung Targeting Peptides
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Cyclic thiolated lung targeting peptides (LTPs) were synthesized as above using 2-chlorotrityl resin as solid support. Cleavage of fully-protected peptide fragments were accomplished under mildly acidic conditions followed by head to tail cyclization using 3- (diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) in Dimethylformamide/Dichloromethane (DMF/DCM) for 5 days at room temperature. Final cleavage and deprotection of the cyclized LTP peptides using Trifluoroacetic acid: Triisopropylsilane:H2O (TFA:TIPS:H2O-90:25:25) was followed by precipitation in Diethyl Ether (EtO2) to isolate the crude peptide. The epsilon amino group of Lysine was then thiolated with 2-iminothiolane (Traut’s reagent) in 0.1M Triethylammonium bicarbonate (TEAbc)/acetonitrile at pH 8.5. The resulting thiolated cyclic LTP peptide was then directly purified by preparative C-18 RP-HPLC on a Waters Delta Prep 4000 chromatography system using standard Acetonitrile/0.1%TFA gradient conditions followed by lyophilization. MALDI- TOF analysis of the purified conjugates on an Applied Biosystems Voyager workstation using α- Cyano-4-hydroxycinnamic acid (CHCA) matrix allowed for confirmation of the expected mass and identity of the final product.
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5
Synthesis and Characterization of PNA Oligomers
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[2,2':6',2''-terpyridine]-4'-carboxylic acid was purchased from Alfa Aesar and used without further purification. The PNA oligomers were prepared by solidphase peptide synthesis on an MBHA resin (Peptides International) downloaded with l-lysine to an 0.1 meq./g NH 2 content. Boc/Z-protected PNA monomers were purchased from PolyOrg and used without further purification. The PNA oligomers were cleaved from the solid support using a mixture of m-cresol/thioanisole/TFMSA/ TFA (1:1:2:6) for 1 h. Cleaved PNA was precipitated using diethyl ether and purified by reversed-phase HPLC using a C18 silica column on a Waters 600. Absorbance was measured with a Waters 2996 photodiode array detector. Characterization of the oligomers was done by MALDI-ToF mass spectrometry using α-cyano-4hydroxycinnamic acid matrix (10 mg/ mL in 1:1 water/acetonitrile, 0.1% TFA). An Applied Biosystems Vo yager workstation with delayed extraction was used for MALDI-ToF mass spectrometry. The calculated and experimental values of m/z for [PNA + H + ] species are provided in Table 2.
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