Peptide lysis analyzerThe operation and use of the equipment cover five core links: equipment calibration and preparation, reagent and sample processing, cracking reaction execution, product purification, and equipment maintenance,Peptide lysis analyzerThe specific operation steps and precautions are as follows:
1、 Equipment calibration and preparation
Component integrity check
Before starting up, check whether the power cord, reactor, pump, valve and other components are intact to ensure that there is no damage or looseness. For example, if the reactor seal ring is found to be aging, it needs to be replaced immediately to avoid gas leakage during the cracking process.
Confirm that the solvent storage bottle is tightly connected to the gas pipeline without any leaks. Check if the compressed air (AIR) pressure is between 60-100psi and the nitrogen (N2) pressure is between 5-10psi.
parameter calibration
Pre set parameters such as cracking temperature, pressure, and flow rate according to experimental requirements. For example, during enzymatic hydrolysis, the temperature needs to be precisely controlled at 37 ℃, with a fluctuation range not exceeding ± 0.5 ℃.
Regularly calibrate temperature sensors (once every six months, with an error controlled within ± 0.2 ℃) and pressure sensors (once a year) to ensure data accuracy.
Equipment preheating
Turn on the power of the synthesizer and preheat the equipment to the set reaction temperature. The preheating time varies depending on the device model and temperature, so patience is required.
2、 Reagent and sample processing
Reagent preparation
Prepare lysis buffer (such as TFA/water/triisopropylsilane=95:2.5:2.5), activation reagents (such as HBTU, DCC), solvents (DMF, DCM), etc., ensure that all materials are fresh and pure, and avoid introducing impurities.
Confirm that the reagent has no precipitation or deterioration. It is recommended to filter the reagent before use and label the opening date. Expired reagents should be discarded in a timely manner.
Sample pretreatment
Pre treat samples with high viscosity or particulate matter by centrifugation, filtration, etc. to prevent blockage of the cracking chamber or damage to the pump body. For example, untreated samples may lead to a 40% increase in pump body wear rate.
3、 Cracking reaction execution
Resin and amino acid fixation
Select a suitable solid phase carrier (such as Rink Amide Resin), swell the resin with dichloromethane (DCM), remove impurities, and drain for later use.
Mix the target amino acid with an activating reagent to form an active ester, which is then fed into the reactor and binds to the deprotected amino group to form peptide bonds.
Cracking reaction
Add pre cooled cracking solution (such as TFA mixture) and stir at room temperature for 2 hours. Real time monitoring of temperature, pressure and other parameters is required during the cracking process, and any abnormalities should be promptly stopped for inspection.
After the cracking is completed, release the reaction solution, filter and remove the resin, wash the resin three times with solvents such as TFA, and combine the filtrate.
4、 Product purification
Concentration and precipitation
Transfer the lysate to a rotary evaporator and concentrate it to a small volume at room temperature. Add methyl tert butyl ether (MTBE) to precipitate the peptide.
Collect the precipitate by centrifugation (10000 rpm, 10 minutes), wash it three times with cold ether, and dry to obtain the crude peptide.
Purification by High Performance Liquid Chromatography (HPLC)
Purification was performed using a C18 reverse phase column, with mobile phase A consisting of 0.1% TFA/water and mobile phase B consisting of 0.1% TFA/acetonitrile. Under gradient elution conditions (phase B increases from 10% to 60% within 0-30 minutes), collect the target peak.
After purification by HPLC, the purity of the peptide can be increased from 85% to 98%.
5、 Equipment maintenance and upkeep
Daily cleaning
After each use, rinse the inner wall of the reactor with deionized water to remove residual lysate and protein fragments. For stubborn stains, soak them in enzyme containing detergent and scrub them.
Perform weekly cyclic disinfection of pipelines with 75% ethanol or 0.1% sodium hypochlorite solution to prevent microbial growth. After disinfection, the pipeline needs to be dried with nitrogen gas.
Replacement of vulnerable parts
Check the elasticity of the sealing ring every 3 months, and replace it immediately if hardening or cracking occurs. Replace the intake filter every 1-2 months to prevent dust from entering the interior of the equipment.
Add specialized lubricating oil (such as silicon-based grease) to the pump body, valves, and other moving parts every quarter to reduce friction and wear.
Long term storage management
When not in use, store the device in a dry, ventilated, and dark place, avoiding direct sunlight and humid environments. Ensure that the equipment is kept away from corrosive gases and liquids.
Apply anti rust oil (such as Vaseline) to the metal surface of the equipment and cover it with a dust cover.