Ultra-performance liquid chromatography is established technology for accurate analysis of IgG n-glycan Due to its sensitivity seems to be and it has ability to provide a specific information of glycan This method is easy to use and has number of different relatively low cost of increments, high reproducibility The ability to glycan isomers. The measurements of protein in plasma could be attractive. in medicine.
also associated with IgG in glycose base and Such and biologic containing the population. You know that of impure state is used to test IgG in glycanes. Actually, it can test n-glycanes of protein.
This is needed in a cross-bound protein of two pure state can be applied to the Protein The OR experimental process is relatively simple while the experimental process need to be standardized. The end-to-end experimental process takes three days to operate, and the experimental steps need to be memorized in order to master the experimental skills. To prepare the samples, thaw the frozen plasma sample.
Then, centrifuge at 80 times G for 10 minutes. Leave the protein G monolithic plate at room temperature for 30 minutes. Transfer 100 micrometers of sample into each well of the two-milliliter collection plate.
Add ultra pure water as one control sample. Dilute the samples with one X PBS by one to seven volume by volume ratio. Clean a 0.45 micron hydrophilic polypropolene filter plate with 200 microliters of ultra pure water.
Repeat the cleaning two additional times. Transfer the diluted samples into the filter plate, and filter the samples into the collective plate using a vacuum pump with pressure at 266.6 to 399.9 Pascals. To prepare the protein G monolithic plates, first discard the storage buffer.
Clean the monolithic plates with two milliliters of ultra pure water, two milliliters of one X PBS, one milliliter of 0.17 molar formic acid, two milliliters of 10 X PBS, and two milliliters of one X PBS sequentially. Remove the following liquid using a vacuum pump. Using a multichannel pipet, transfer the filtered samples to the protein G monolithic plate for IgG binding and cleaning.
Then, add two milliliters of one X PBS to clean the monolithic plates. Remove the PBS using a vacuum pump, and repeat the cleaning two additional times. Illute IgG with one milliliter of 0.17 molar formic acid, and filter the samples into the collection plate by vacuum pump.
Then, add 170 microliters of one molar ammonium bicarbonate into the collection plate. Detect the IgG concentration using an absorption spectropetometer at the optimal wavelength of 280 nanometers. Place the extracted IgG in an oven to dry at 60 degrees Celsius for three hours.
Determine the amount to be observed according to its concentration as described in the manuscript, and preserve it in minus 80 degrees Celsius. Gather the dried IgG, and store chemicals including 1.33%SDS, four percent IgePal and 5%PBS at room temperature. Prepare indoglycosidase F enzyme by diluting 250 units of enzyme with 250 microliters of ultra pure water.
To denature the IgG, add 30 microliters of 1.33%SDS and mix by vortexing. Transfer the sample into a 65 degree Celsius oven for 10 minutes. Then, remove it from the oven, and let it cool for 15 minutes.
Add 10 microliters of four percent IgePal into the same sample, and place it on a shaking incubator for five minutes. Add 20 microliters of five X PBS and 30 to 35 microliters of 0.1 molar sodium hydroxide to regulate the pH to 8.0, and mix by vortexing. Add four microliters of endoglycosidase F enzyme, and mix by vortexing.
Then, incubate for 18 to 20 hours in a 37 degrees Celsius water bath. The next day, place the released gylcans in an oven at 60 degrees Celsius to dry for two and a half to three hours. Save the release glycans at minus 80 degrees Celsius until further measurement.
Prepare the two amino benzimide labeling reagent with 24.5 microliters of DMSO, 10.5 microliters of acetic acid, 0.7 milligrams of two amino benzimide, and six milligrams of sodium cyanoborohydride. Next, in a dark room, label the glycans by adding 35 microliters of two amino benzimide labeling reagent. Transfer the labeled glcans to an oscillator for five minutes, and then, transfer it to the oven for three hours at 65 degrees Celsius.
After that, transfer it to room temperature for 30 minutes. The IgG n-glycase, we'll label it with in order to be detected by the fluorescent detect of impure state. Pretreat a 0.2 micron GHP filter plate with 200 microliters of 70%ethanol, 200 microliters of ultra pure water, and 200 microliters of 96%aceto nitrile at four degrees Celsius.
Then, remove the waste using a vacuum pump. To purify the two amino benzimide labeled glycans, add 700 microliters of 100%acetonitrile to the sample, and transfer it to a shaking incubator for five minutes. Centrifuge at 134 times G for five minutes at four degrees Celsius.
Transfer the supernatent to a 0.2 microns GHP filter plate for two minutes, and remove the filtrate using the vacuum. Wash the two amino benzimide labeled glycan with 200 microliters of 96%acetonitrile at four degrees Celsius, and remove the filtrate using a vacuum pump five to six times. Illute two amino benzimide labeled glycan with 100 microliters of ultra pure water three times.
Transfer the two amino benzimide labeled glycan into an oven to dry at 60 degrees Celsius for three and a half hours. Save the labeled n-glycans at minus 80 degrees Celsius until further measurement. First, open the software to control the mobile phases.
Wash UPLC instruments with 50%solvent B and 50%solvent C at the flow rate of 0.2 milliliters per minute for 30 minutes. Then, wash with 25%solvent A and 75%solvent B at a flow rate of 0.2 milliliters per minute for 20 minutes. Then, add a flow rate of 0.4 milliliters per minute.
Dissolve the labeled n-glycans with 25 microliters of a mixture of 100%acetonitrile and ultra pure water at a two to one volume by volume ratio. Then, centrifuge at 134 times G for five minutes at four degrees Celsius, and use a dry pipe to load 10 microliters of the supernatent into the UPLC instruments. Separate the labeled n-glycans at flow rate of 0.4 milliliters per minute with a linear gradient of 75%to 62%acetonitrile for 25 minutes.
Then, perform an analytic run by Dexteran calibration ladder glycopeptide column on a UPLC at 60 degrees Celsius. Detect n-glycan fluorescents at X citation and emission wavelengths of 330 nanometers and 420 nanometers, respectively. As shown in this figure, IgG n-glycans were analyzed into 24 initial IgG glycan peaks based on peak position and retention time.
The n-glycan structures are available in 24 types through mass spectrometry detection. To asses the repeatability and stability of the method, the standard sample was tested in parallel six times. The glycan peaks with relatively small proportions showed high measurement errors of more than 10%of coefficient of variation.
In the piece and important to forming to an established of IgG n-glycan structure, especially for establishing terminal Therefore, instead of 3.3 IgG n-glycase found in is critical in this the importance of IgG. Glycans from are known to be different. With the developments in glycan prototomics, combined in glycans to explore information to this such that It can be used as a potential for today's diagnosis.
After this technique development, it provides a new way to explore the
