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Method for utilizing affinity column to analyze holoprotein

A whole protein and affinity technology, applied in the field of proteomics, can solve the problems of heavy workload, long operation time, time-consuming and laborious, and achieve the effect of increasing the number of proteins, simplifying the complexity, and being easy to automate.

Inactive Publication Date: 2010-04-14
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in actual operation, large-scale removal of high-abundance proteins, traditional multidimensional liquid chromatography and commonly used multidimensional electrophoresis pre-separation methods are complicated to operate, time-consuming, laborious, and heavy workload, which is not conducive to hydrophobic proteins, extremely small and extremely small molecular weight proteins, extremely Analysis of high and very low isoelectric point proteins and membrane proteins
Moreover, the large-scale removal of high-abundance proteins needs to first analyze and identify high-abundance proteins, and then prepare corresponding protein antibodies. The operation time is long, the workload is heavy, and the operation is cumbersome.

Method used

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  • Method for utilizing affinity column to analyze holoprotein
  • Method for utilizing affinity column to analyze holoprotein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Synthesis of affinity materials based on epichlorohydrin

[0030] Take Sepharose CL-4B (100 grams) and wash it with 10 times the volume of deionized water, drain it into a wet cake; then suspend it in 50ml activation buffer (1M NaOH, 2.5g sodium borohydride, 10ml epichlorohydrin), React at a constant temperature of 60°C for 2 hours under stirring, stop the reaction when the pH is close to 7.0, then pour it into a glass frosted funnel, wash with 10 times the volume of distilled water under suction filtration, and distribute the Sepharose 4B activated by epichlorohydrin in a can In a sealed glass bottle (20g / bottle).

[0031] Take 0.5g of various amino compounds (see Table 1) and dissolve them in 25mL of 0.1M sodium hydroxide / L dioxane, respectively add them to the bottles containing activated Sepharose 4B, label them well, and put them under stirring at 60 ℃ reaction 24h. Add 1mL of mercaptoethanol, continue to react for 2 hours, fully wash with 10 times the volume of ...

Embodiment 2

[0077] Synthesis of Biomimetic Ligands Using Trichlorotriazazine as Skeleton

[0078] Suspend 100 grams of epichlorohydrin-activated Sepharose CL 4B medium in 350 ml of deionized water, then add 150 ml of 35% (v / v) ammonia water, and keep the temperature at 30° C. for 12 hours under stirring (200 r / min). NH 2 - Suspend SepharoseCL-4 B in 350ml of 50% (v / v) ice-bathed acetone solution, then dissolve 8g of trichlorotriazoxide in 80ml of -20°C pre-cooled acetone, quickly add the medium suspension, wash with saturated NaHCO 3 Keep the pH between 6.5 and 8.0, and continue stirring at 0 to 4°C for 2 hours to stop the reaction.

[0079] Use 3 times of medium volume of acetone, acetone: deionized water (volume ratio 1: 1), deionized water, wash the reaction in sequence to obtain dichlorotriazine-amino-Sepharose CL 4B; weigh 20g of dichlorotriazoxide -Amino-Sepharose CL 4B, mixed with a 5-fold molar excess of amino compound (R1) dissolved in a certain amount of distilled water (20-50...

Embodiment 3

[0081] Protein adsorption performance evaluation and selection

[0082] Evaluate the adsorption protein performance of the affinity separation material library, and select an affinity column whose adsorption protein species account for 20 to 80% of the total protein.

[0083] First, take 1ml of the synthesized ligand medium and fill it into the chromatography column respectively, write the label, and fully wash the balance with 15ml of equilibrium buffer (pH7.0, 10mM phosphate buffer containing 0.1M NaCl); then take Load 1-4mg tissue protein, combine on the column for 30 minutes, wash off unbound protein with 10-20ml equilibration buffer (monitored by protein UV detector until the baseline is flat); finally use 3ml elution buffer (pH12, 10mM Glycine-NaOH buffer) to elute the bound protein on the column and immediately adjust the pH to neutral. The collected protein samples bound by each ligand were analyzed by SDS-PAGE electrophoresis, and the ligand media with large differen...

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Abstract

A method for utilizing an affinity column to analyze holoprotein in the technical field of protein group comprises the following steps: compounding affinity separating materials, obtaining an affinity separating material base, extracting biology sample holoprotein, screening affinity material combinations whose absorbing protein category accounts for 20 to 80% of holoprotein from the affinity separating material base obtained from the first step, selecting one or a plurality of affinity material combinations from the affinity material combinations obtained from the second step for respectively filling affinity columns, then, utilizing the obtained affinity columns, adopting a cascade combination analysis method, a series combination analysis method, or a cascade and series mixed combination method to analyze biology sample holoprotein, obtaining a plurality of groups of samples whose protein complexity is simplified, respectively carrying out parenzyme digestion and LC-MS / MS analysis to the samples obtained from the third step, and thereby finishing the holoprotein analysis of biology sample holoprotein. The method of the invention can overcome the technical defect that high abundance protein submerges low abundance protein signals, and improves the extremity property protein detectable rate.

Description

technical field [0001] The invention relates to a method in the technical field of proteome, in particular to a method for analyzing whole protein by using an affinity column. Background technique [0002] Proteins are molecular carriers that perform biological functions in organisms. The analysis and identification of whole proteins in biological samples plays an important role in elucidating biological functions, physiological states, and the development and treatment of diseases. Biological samples often contain thousands of proteins. Generally, no more than twenty high-abundance proteins account for 99% of the total protein, while thousands of low-abundance proteins account for less than 1% of the total protein. The abundance range of high-abundance proteins and low-abundance proteins is as high as 9 orders of magnitude. Biomass spectrometry based on shot-gun strategy can quickly and easily identify various proteins in biological samples. However, due to the limitatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/60B01J20/286
Inventor 李荣秀谭青乔
Owner SHANGHAI JIAO TONG UNIV
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