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A rapid method for the determination of protein content by removing interference

A protein and protein precipitation technology, applied in the field of sucrose to promote protein precipitation, can solve problems such as affecting the detection results, and achieve the effects of high detection sensitivity, short precipitation time, and high recovery rate

Active Publication Date: 2018-05-29
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, trichloroacetic acid can react with Coomassie Brilliant Blue G250, which affects the detection results.

Method used

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  • A rapid method for the determination of protein content by removing interference
  • A rapid method for the determination of protein content by removing interference
  • A rapid method for the determination of protein content by removing interference

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1 Acetone precipitation removes polysorbate 80 (Tween 80):

[0055] 1) Sample preparation: Add appropriate Tween 80 solution into bovine serum albumin (BSA) solution, so that the final concentration of Tween 80 solution is 0, 0.5, 2 mg / ml, and the final concentration of BSA is 20 μg / ml. Take 0.6ml of each into 2ml EP tubes and pre-cool them.

[0056] 2) Precipitate protein: add 1.4ml pre-cooled acetone to each sample solution, shake well, place in ice bath for 1h, centrifuge at 12000g for 15min to remove supernatant; Place in the bath for 10 minutes, centrifuge at 12000g for 10 minutes to remove the supernatant, and repeat once more. Blow dry naturally to remove acetone residue. Finally, 0.6 ml of water was added to the protein pellet.

[0057] 3) Spectrum scanning: Take 0.6ml of BSA solution containing 0, 0.5, 2mg / ml Tween 80, and each sample after precipitation, add 0.15ml Coomassie Brilliant Blue G250 dye solution (purchased from BioRad Company), mix an...

Embodiment 2

[0059] Example 2 protein standard curve

[0060] 1) Standard product preparation: Prepare BSA standard protein with water to 6 concentrations of 0, 2.5, 5, 10, 15, and 20 μg / ml, and prepare 2 parallel tubes, 0.6 ml in each tube.

[0061] 2) Color development: Add 0.15ml of Coomassie Brilliant Blue G250 dye solution (purchased from BioRad) and mix well, let stand for 10min, mix well, pipette 0.2ml into the microtiter plate, duplicate wells 3 times, measure the OD value at 595nm .

[0062] 3) Linear regression analysis: linear regression analysis was performed with the concentration of BSA as the ordinate and the OD value as the abscissa to obtain the regression equation of the standard curve.

[0063] 4) See the result figure 2 , in the range of 0-20μg / ml, the protein concentration has a linear relationship with the OD value, and the correlation coefficient R 2 >0.99.

Embodiment 3

[0064] The impact of the sucrose of embodiment 3 different concentrations on acetone precipitation protein

[0065] 1) Sample preparation: BSA solutions containing 0, 10, 50, and 100 mg / ml sucrose were prepared, the final concentration of BSA was 20 μg / ml, and 0.6 ml of each was put into a 2 ml EP tube.

[0066] 2) Precipitate protein: Add 1.4ml of pre-cooled acetone and shake well, place in ice bath for 15min, 30min, 60min respectively, centrifuge at 12000g for 15min to remove supernatant; add 0.6ml of pre-cooled acetone to each tube, mix well, place in ice bath 10min, centrifuge at 12000g for 10min to remove the supernatant, repeat once. Blow dry naturally to remove acetone residue.

[0067] 3) Protein standard curve: same as Example 2.

[0068] 4) Determination of protein content: add 0.6 ml of aqueous solution to the sample after precipitation, mix well, and add 0.15 ml of Coomassie Brilliant Blue G250 dye solution (purchased from BioRad Company) to each sample. The OD ...

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Abstract

The invention discloses a method for promoting protein precipitation by using sucrose, and a method for measuring trace proteins based on the method for quickly removing surfactant interference. The method is characterized in that the protein in the solution is precipitated by adding an organic solvent and sucrose, and the protein is effectively separated from other components such as a surfactant by centrifugation, so as to obtain a protein precipitate from which other components such as a surfactant are removed. The protein precipitation can not only be used to determine its protein content by the Coomassie Brilliant Blue G250 method, but also can be used for SDS‑PAGE or HPLC detection. This method is especially suitable for protein quantification of protein drugs containing surfactants. The invention has the characteristics of high protein recovery rate, high detection sensitivity, simple operation steps, rapid detection and the like.

Description

technical field [0001] The invention relates to a method for promoting protein precipitation by using sucrose, and a method for determining protein content based on the method for quickly removing interference from interfering substances such as surfactants. Background technique [0002] Because protein drugs have the advantages of specific targets and obvious curative effects, the research on protein drugs has attracted more and more attention. In recent years, a variety of recombinant protein drugs have been approved by the State Food and Drug Administration for clinical treatment of various diseases. . However, the protein itself has the characteristics of being easily degraded and aggregated and inactivated, so how to maintain its activity for a long time has always been a difficulty in the field of biopharmaceuticals. In order not to inactivate protein drugs, it is usually necessary to add some non-protein excipients (such as sucrose, mannitol, lactose, etc.) or nonion...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/28G01N21/31G01N21/33G01N33/68
Inventor 田志刚郑晓东程永凤魏海明刘玉玲孙汭
Owner UNIV OF SCI & TECH OF CHINA
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