Method for detecting protein content

A protein content and detection method technology, applied in the field of protein content detection, can solve the problems of the influence of the absorbance value of protein samples, inconvenient protein content determination, poor tolerance of surfactants, etc., and achieves low measurement cost, high speed and good repeatability. Effect

Inactive Publication Date: 2011-11-23
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]The Lowry method is cumbersome to operate and is also affected by many chemical components, such as when the content of surfactant Triton X-100 or SDS is only 1%, or metal ions When the concentration of chelating agent EDTA reaches 10 mM, or the content of urea reaches 3 M, or the concentration of ammonium sulfate reaches 0.25 M, etc., it will seriously interfere with the detection results; although the BCA method is less affected by the interference of surfactants, it is not affected by reducing agents and metal ion chelation. Sensitive to the prese

Method used

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  • Method for detecting protein content
  • Method for detecting protein content
  • Method for detecting protein content

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] [Example 1] BSA standard curve and chromogenic product stability experiment with wavelengths of 570, 595 and 630 nm

[0051] 1) Preparation of standard samples

[0052] Weigh bovine serum albumin (BSA) and dissolve it in distilled water to adjust the final concentration to 4 mg / mL, which is the prepared standard sample.

[0053] 2) Reagent preparation

[0054] Reagent ①:

[0055] Sodium carbonate 1.89 mol / L

[0056] Sodium hydroxide 1.00 mol / L

[0057] Reagent ②:

[0058] Sodium tartrate 0.25 %

[0059] Copper sulfate 0.125 %

[0060] SDS 2.5%

[0061] When preparing, it is necessary to mix sodium tartrate and copper sulfate first and then add SDS.

[0062] When in use, reagent ① and reagent ② are mixed at a ratio of 50 / 1 and prepared for immediate use.

[0063] Reagent ③:

[0064] Dilute the 2 N Folinol solution, then adjust the pH to 1, and finally make it 10 times the volume.

[0065] 3) Preparation of protein concentration st...

Embodiment 2

[0070] [Example 2] Determination of protein content in mammalian cells under surfactant interference

[0071] 1) Preparation of standard samples

[0072] Weigh bovine serum albumin (BSA) and dissolve it in distilled water to adjust the final concentration to 4 mg / mL, which is the prepared standard sample.

[0073] 2) Reagent preparation

[0074] Reagent ①

[0075] Sodium carbonate 1.89 mol / L

[0076] Sodium hydroxide 1.00 mol / L

[0077] Reagent ②

[0078] Sodium tartrate 0.25 %

[0079] Copper sulfate 0.125 %

[0080] SDS 2.5%

[0081] When preparing, it is necessary to mix sodium tartrate and copper sulfate first and then add SDS.

[0082] When in use, reagent ① and reagent ② are mixed at a ratio of 50 / 1 and prepared for immediate use.

[0083] Reagent ③

[0084] Dilute the 2 N Folinol solution, then adjust the pH to 1, and finally make it 10 times the volume.

[0085] 3) Mammalian cell culture and cell protein extraction

[0086] will be 1.0X10 6 Human pr...

Embodiment 3

[0097] [Example 3] Determination of mammalian cell protein content under reducing agent interference

[0098] 1) Preparation of standard samples

[0099] Weigh bovine serum albumin (BSA) and dissolve it in distilled water to adjust the final concentration to 4 mg / mL, which is the prepared standard sample.

[0100] 2) Reagent preparation

[0101] Reagent ①

[0102] Sodium carbonate 1.89 mol / L

[0103] Sodium hydroxide 1.00 mol / L

[0104] Reagent ②

[0105] Sodium tartrate 0.25 %

[0106] Copper sulfate 0.125 %

[0107] SDS 2.5%

[0108] When preparing, it is necessary to mix sodium tartrate and copper sulfate first and then add SDS.

[0109] When in use, reagent ① and reagent ② are mixed at a ratio of 50 / 1 and prepared for immediate use.

[0110] Reagent ③

[0111] Dilute the 2 N Folinol solution, then adjust the pH to 1, and finally make it 10 times the volume.

[0112] 3) Mammalian cell culture and whole protein extraction

[0113] will be 1.0X10 6 Human prostate ...

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Abstract

The invention relates to a method for detecting protein content. The method comprises the following steps of: preparing standard protein samples and reagents (1), (2) and (3); preparing the standard protein samples with gradient; adding the reagents (1), (2) and (3) into the gradient samples and samples to be tested by a micropore method or a standard test tube method; measuring absorbance; drawing a standard curve; and calculating the concentration according to the absorbance of the samples to be tested. By the method, the protein content can be detected accurately. The method is applicable to protein solution containing a plurality of interfering substances. By the method, the inclusiveness for the interfering substances such as a surfactant, a reducing agent, a chelating agent, a nitrogen-containing compound and the like in the protein analysis and detection process can be improved. The method is a simple, quick, high-sensitivity and high-stability method for detecting the protein concentration and has a wide application prospect in the field of life science research.

Description

technical field [0001] The invention belongs to the technical field of biochemical analysis and detection, and in particular relates to a protein content detection method. Background technique [0002] Conventional protein concentration detection methods mainly include Lowry method, BCA (Bicinchoninic acid) method and Bradford method. The Lowry method is mainly based on the Cu under alkaline conditions 2+ Form a blue complex with the -CO-NH- group of the peptide backbone, and then reduce the phosphotungstic acid and phosphomolybdic acid in the Folin's phenol reagent to tungsten and molybdenum blue for further color development. The BCA method is to use BCA to react with the Cu formed in the first step of the Lowry method. + It is measured by the blue-purple complex formed by binding. The basis of the Bradford method is that the dye Coomassie Brilliant Blue G-250 can combine with lysine and arginine in proteins or peptides with a molecular weight above 3 kD to form colored...

Claims

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Application Information

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IPC IPC(8): G01N21/31G01N33/68
Inventor 卢山董源
Owner NANJING NORMAL UNIVERSITY
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