Parent peptide t-DPH1 and its designed analogues were synthesised using a Tribute peptide synthesiser (Protein Technologies, Tucson, AZ, USA). The products were purified by reverse-phase HPLC (Phenomenex Aeris PEPTIDE 5 µm XB-C18 column, 250 × 21.2 mm, Macclesfield, Cheshire, UK) with a linear gradient formed from 80% buffer A (0.05/99.5 (v/v) TFA/water) and 20% buffer B (0.05/19.95/80.00 (v/v/v) TFA/water/acetonitrile) to 0% buffer A: 100% and buffer B in 60 min at a flow rate of 8 mL/min. The masses of purified products were verified by MALDI-TOF MS (matrix-assisted laser dissociation ionised-time of flight mass spectrometry) (Voyager DE, Perspective Biosystem, Foster City, CA, USA) in positive detection mode using CHCA (α-cyano-4-hydroxycinnamic acid) as the matrix.
Tribute peptide synthesiser
Manufactured by Protein Technologies
Sourced in United States
The Tribute Peptide Synthesiser is a laboratory instrument designed for the automated synthesis of peptides. It is capable of producing a wide range of peptide sequences with high purity and efficiency.
Automatically generated - may contain errors
Lab products found in correlation
Aeris peptide 5 m xb c18 column, by Phenomenex (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Trifluoroacetic acid tfa, by Merck Group (1 mentions)
Ddh2o, by Merck Group (1 mentions)
Diethyl ether et2o, by Merck Group (1 mentions)
1 2 ethanedithiol edt, by Merck Group (1 mentions)
Dichloromethane dcm, by Merck Group (1 mentions)
Alpha 1 2 ldplus freeze dryer, by Martin Christ (1 mentions)
5 protocols using tribute peptide synthesiser
1
Synthesis and Purification of Peptide Analogues
2
Synthesis and Purification of GV30 Peptide
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GV30 and its derivatives were synthesised by using a Tribute Peptide Synthesiser (Protein Technologies, USA). The amino acids and the activator, HBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), were weighted in vials. After coupling, the cleavage solution (94 % trifluoroacetic acid (TFA) + 2 % triisopropylsilane(TIS) + 2 % 1,2-ethanedithiol (EDT) + 2 % H2O) was prepared to perform the cleavage reaction of the protection groups. Then, ice-cold diethyl ether was prepared to wash the peptide, and the washed peptide was then dissolved in buffer A solution (TFA /water (0.05/99.95, v/v)). Finally, 0.3 % H2O2 was prepared as an oxidising agent to form disulphide bonds.
3
Synthetic Peptide Production Protocol
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The natural peptide discovered, as well as its analogues, were chemically-synthesised using a Tribute Peptide Synthesiser (Protein Technologies, Tucson, AZ, USA). Peptides were synthesised from C-terminal end to N-terminal end as previous study [17 (link)]. The Fmoc protecting groups were deprotected using 20% (v/v) piperidine in DMF, and each amino acid residue was activated and coupled using 11% (v/v) NMM in 89% (v/v) DMF combined with activator HBTU. The synthetic peptides were purified by RP-HPLC and lyophilized for functional tests.
4
Fmoc-Based Peptide Synthesis Protocol
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Fmoc-chemistry peptide synthesis was performed for all peptides in this study, using a Tribute Peptide Synthesiser (Protein Technologies, Tucson, AZ, USA), which was described in the previous study [53 (link)]. Briefly, 0.3 mmol of each Fmoc amino acid was weighted and mixed with an equal amount of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (hexafluorophosphate benzotriazole tetramethyl uronium, HBTU) in the loading vial. Rink amide resin (250 mg) was employed as the solid phase for the synthesis of peptide chain as well as providing the C-terminal amide for all peptides. In the synthesis process, the peptide bonds were coupled in the presence of HBTU that was dissolved by 1 M N-methylmorpholine (NMM) in dimethylformamide (DMF), followed by the deprotection of α-NH2 by 20% (v/v) piperidine in DMF. Once the synthesis was accomplished, a 25 mL cleavage cocktail (trifluoroacetic acid (TFA)/water/thioanisole/1,2-Ethanedithiol = 94/2/2/2 (v/v/v/v)) was added to the resin–peptide matrix for 2–4 h at room temperature for releasing the peptide chains from the resin as well as deblocking the side chains. The synthetic peptides were purified by reverse-phase HPLC and lyophilized for functional tests.
5
Peptide Synthesis and Purification Protocol
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A Tribute peptide synthesiser (Protein Technologies, Inc, USA) was utilised to synthesise the mature peptide. After synthesis, the peptide was transferred to a 50-ml round-bottomed flask. Twenty-five ml per gram of mixture cleavage solution (94 % Trifluoroacetic acid (TFA) (Sigma-Aldrich, USA), 2 % dd H2O, 2 % thioanisole (TIS) (Sigma-Aldrich, USA) and 2 % 1–2 ethanedithiol (EDT) (Sigma-Aldrich, USA)) were added into the round-bottomed flask. The round-bottomed flask was stirred with a rotor in the fume hood for 2 h. After cleavage, the mixture solution was filtered by a Buchner funnel, and 3 ml of dichloromethane (DCM) were used to wash (Sigma-Aldrich, USA) three times. Diethyl ether (Et2O) (Aldrich, USA) was added to the mixture to 50 ml in a 50-ml tube. Moreover, the tube was placed at a temperature of −20 °C to precipitate the peptide. After drying in the fume hood, 10 ml of HPLC buffer A (0.5 ml TFA/999.5 ml H2O) were used to dissolve the peptide, and then buffer B (0.5 ml TFA/199.5 ml H2O/800 ml acetonitrile (Sigma-Aldrich, USA)) 5 ml. The tube was put in an Alpha 1–2 LD plus freeze dryer (CHRIST, Germany) for 48–50 h. The dry peptide was then stored at −20 °C, prior to further analyses.
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