RP-HPLC has become an important tool for the separation and analysis of peptides.
It is widely used in the characterization of peptide therapeutic products and the identification of these products and impurities in the biotechnology industry.
Before identification of peptides by mass spectrometry, RP-HPLC plays an important role in the separation of peptides. It has been used for the purification of a variety of proteins and peptides in exploratory research, as well as large-scale purification of protein therapeutic drugs.
General objectives and methods of purification
The peptides from natural or synthetic sources can be used for chromatographic separation after some crude extraction steps (such as homogenization, centrifugation, ammonium sulfate precipitation, etc.).
Then the capture chromatography (capture chromatography) is carried out. The main goal is to concentrate and remove a large number of easily removed impurities. This step Zui is concerned with the flow rate and load, often using high loading and fast flow gels.
Aung Tuo lees biological prompts, concentrated partially purified samples were carried out by intermediate chromatography (intermediate chromatography) to remove impurities that are difficult to remove. Step Zui is concerned with resolution, often using high resolution fine particle gel.
In order to get the Zui final product that meets the requirements, remove residual impurities and target protein polypeptides or degradation fragments, polishing chromatography is used to gel filtration chromatography with high resolution gel filtration.
Preparation before purification
(1) Sample stability test. a. Determine the stability of the sample at pH 2-9; B. determine the stability of the sample in 0-4 mol / L NaCl and 0-2 mol / L ammonium sulfate; C. determine the stability of the sample in 0-50% ethanol and methanol; D. determine the stability of the sample at 4-40 ℃; e. let it stand overnight at room temperature to determine the stability of proteolytic enzyme.
(2) Sample pretreatment. a. Removal of particulate matter from the sample (0.45-0.22 micron membrane filtration or 10000 g centrifugation for 15 minutes)
b. Remove lipids from the sample (10000 g centrifuged for 15 minutes or extracted with organic solvent)
c. Removal of nucleic acids from the sample (digestion with nuclease or precipitation of nucleic acids)
d. Inhibition of proteolytic enzymes in samples (addition of protease inhibitors, rapid first step separation at low temperature or expression of recombinant proteins in protease deficient hosts)
(3) Storage conditions: short term storage (less than 24 hours)
a. Avoid approaching or reading the stable Ji limit of the sample to prevent protein denaturation or precipitation B. refrigerate in a closed container for a long time (several days)
a. B. same as above C. add appropriate Jun inhibitor for long-term storage A. ditto B. freeze or freeze-drying (vacuum freeze-drying) preservation.
Establishment of evaluation methods for each purification step
a. Objective to determine the peptide content, enzyme activity, biological activity, radioimmunoassay, enzyme-linked immunosorbent assay, immunoelectrophoresis and fluorescence.
b. The methods used in the determination of total protein content were UV absorption method, Lowry method, Bradford dye binding method (Coomassie brilliant blue G-250), etc.
C and sample complexity were detected by HPLC (ion exchange, gel filtration, reversed-phase), SDS-PAGE, isoelectric focusing and capillary electrophoresis (CE).
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