CN111537316A - Amino black staining solution, kit and method for detecting protein content - Google Patents
Amino black staining solution, kit and method for detecting protein content Download PDFInfo
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 title claims abstract description 43
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- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
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- DHRLEVQXOMLTIM-UHFFFAOYSA-N phosphoric acid;trioxomolybdenum Chemical compound O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.OP(O)(O)=O DHRLEVQXOMLTIM-UHFFFAOYSA-N 0.000 description 1
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/44—Sample treatment involving radiation, e.g. heat
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
Abstract
The invention is suitable for the technical field of protein quantification, and provides amino black staining solution, a kit and a method for detecting protein content, wherein the kit comprises the amino black staining solution, rinsing solution, eluent and a plurality of groups of standard protein solutions with different concentrations; wherein the amino black staining solution comprises methanol, glacial acetic acid, amino black and water. The kit containing the amino black staining solution provided by the invention fully utilizes the fact that amino groups and carboxyl groups of amino acids in protein can react in a proper acid-base environment, so that the protein is positively charged, the amino black appears in a negative ion form, and a large number of dye molecules are deposited on the surface of the protein under the interaction of positive and negative charges, so that protein traces are shown in dye colors, and further, the protein quantification method which is simple in operation, high in sensitivity and good in accuracy can be provided.
Description
Technical Field
The invention belongs to the technical field of protein quantification, and particularly relates to amino black staining solution, a kit and a method for detecting protein content.
Background
Protein quantification results are fundamental data in biomedical research. Whether it is accurate or not is related to the reliability of the research result. Protein quantification is a precondition work which needs to be done before many biomedical experiments are done, and particularly when Western Blot is done, the sample loading amount can be determined only by determining the protein content in a sample to be tested, so that a foundation can be laid for further experiments.
The total protein quantitative technical method of the sample has more limiting conditions, complex operation and narrow application range, and at present, the method for measuring the protein content in the experiment comprises the following steps: lowry method, BCA method, Bradford method, sulfosalicylic acid precipitation method, etc. The determination principle is as follows: the measurement principle of the Lowry method is that protein and basic copper sulfate react to form a copper-peptide bond complex, and the copper-peptide bond complex, tryptophan and tyrosine together act to reduce phosphomolybdic acid and phosphotungstic acid in a phenol reagent to generate a blue compound; the BCA method is based on the principle that protein valence copper ions are reduced into cuprous ions, and the cuprous ions are combined with BCA in an alkaline solution to generate a purple red complex. The Lowry method and the BCA method are chemical methods. The sulfosalicylic acid precipitation method belongs to a physical method, and the Bradford method belongs to a dye binding method, namely Coomassie brilliant blue G-250 is reddish brown in an acid solution, is combined with protein and then turns into blue, and the color intensity is in a direct proportion relation with the protein concentration.
However, these methods have some disadvantages, such as some amino acids, NH, which are the main factors affecting the measurement of proteins by Lowry method+4A facultative ionic buffer solution, a nonionic surfactant, sucrose and a sulfur-containing compound; the main factors influencing the protein detection by the BCA method are sucrose and NH+4Although the precipitation method of urea, EDTA and sulfosalicylic acid overcomes the above factors, the precipitation properties of different proteins are very different, so that the protein content is determined by the method by selecting the same or similar proteins as standards, the main factors influencing the Bradford method are glycerol, acetic acid, detergent and some alkaline buffer systems, the BCA method is more commonly used in experiments, and the denatured solution and the renatured solution of inclusion bodies often contain urea and β -mercaptoethanol which interfere with the determination of the BCA method, so the BCA method is not suitable for the determination of the protein content in the denatured solution containing urea and β -mercaptoethanolHowever, in the Western blotting experiment, a Loading Buffer containing β -mercaptoethanol is required, so that the measurement by the BCA method is not accurate enough.
Therefore, there is a need to find a method for more accurately determining the protein content in a sample to avoid the experimental error easily generated.
Disclosure of Invention
The embodiment of the invention aims to provide an amino black staining solution for detecting protein content, and aims to solve the problems in the background art.
The embodiment of the invention is realized in such a way that the amino black staining solution for detecting the protein content comprises the following components in each liter: 200-400 mL of methanol, 50-150 mL of glacial acetic acid, 0.05-0.15 g of amino black and the balance of water.
As a preferable scheme of the embodiment of the invention, each liter of the amino black dyeing liquid comprises the following components: 250-350 mL of methanol, 80-120 mL of glacial acetic acid, 0.08-0.12 g of amino black and the balance of water.
Another objective of the embodiments of the present invention is to provide a kit for detecting protein content, which includes the amino black staining solution.
As another preferable scheme of the embodiment of the invention, the kit also comprises a rinsing solution, an eluent and a plurality of groups of standard protein solutions with different concentrations.
In another preferred embodiment of the present invention, the standard protein solution is a standard bovine serum albumin solution.
As another preferable scheme of the embodiment of the invention, the rinsing liquid is a methanol aqueous solution, and the volume concentration of the methanol aqueous solution is 40-60%.
In another preferred embodiment of the present invention, the eluent is a sodium hydroxide solution, and the molar concentration of the sodium hydroxide solution is 0.5-1.5 mol/L.
Another object of the embodiments of the present invention is to provide a method for detecting protein content, which is performed by using the above kit, and specifically includes the following steps:
respectively detecting a plurality of groups of standard protein solutions with different concentrations, and drawing a standard curve;
after carrying out denaturation treatment on a protein sample to be detected, placing the protein sample on a carrier, and rinsing the protein sample by using the rinsing liquid;
after the color of the rinsed protein sample to be detected disappears, placing the carrier in the amino black staining solution for staining;
after the color of the dyed protein sample to be detected is stable, placing the carrier in the rinsing liquid for rinsing; then, taking out the protein sample to be detected on the carrier, and placing the protein sample in the eluent for oscillation treatment to obtain a test solution;
and carrying out absorbance test on the test solution, and calculating to obtain a concentration value of the protein in the protein sample to be tested according to the standard curve.
In another preferable embodiment of the present invention, in the step, the temperature of the denaturation treatment is 93 to 97 ℃.
As another preferable mode of the embodiment of the present invention, the carrier is a nitrocellulose membrane.
According to the amino black staining solution for detecting the protein content, provided by the embodiment of the invention, the amino group and the carboxyl group of amino acid in protein can be fully utilized to react in a proper acid-base environment, so that the protein is positively charged, the amino black appears in a negative ion form, and a large number of dye molecules are deposited on the surface of the protein under the interaction of the positive and negative charges, so that the protein trace is shown in a dye color, and further, the protein quantification method which is simple to operate, high in sensitivity and good in accuracy can be provided.
The kit provided by the embodiment of the invention has the advantages that a large number of dye molecules are deposited on the surface of protein by a physical adsorption dyeing method, so that the protein trace shows the dye color.
In addition, the method for detecting the protein content provided by the embodiment of the invention is used for detecting the denatured protein sample, so that the protein degradation before and after denaturation of the sample in the detection process is avoided, and the inaccuracy of the measurement result can be avoided.
In summary, compared with the prior art, the method for detecting protein content provided by the embodiment of the invention has the following advantages:
(1) the operation is simple, and special instruments and equipment are not needed. The method has no toxic reagent in the experiment, and is very suitable for basic laboratories and primary personnel to operate and use.
(2) The time consumption is short, and the detection efficiency is high. The time consumption of the experimental process is less than half an hour, the detection can be carried out by utilizing a 96-well plate, the double holes are parallel, at least 40 samples can be detected at one time, and the detection efficiency is high.
(3) The detection sensitivity is high, the lower limit of the detected protein concentration can reach 10 mu g/mL, the dosage of a detection sample is less and only 40 mu L or less is needed, and the cost is low.
Drawings
FIG. 1 is a standard graph of the kit provided in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
This embodiment provides a kit for detecting protein content, which comprises amino black staining solution, rinsing solution, eluent and several sets of standard protein solutions with different concentrations. In addition, the kit may further include a petri dish, a punch, a nitrocellulose membrane, etc. required for the detection process, but is not limited thereto.
Wherein, the amino black staining solution is obtained by adding 30mL of methanol, 10mL of glacial acetic acid and 0.1g of amino black into a container, adding water to a constant volume of 100mL, and uniformly mixing; the plurality of groups of standard protein solutions with different concentrations are standard bovine serum albumin solutions, and the concentrations of the standard protein solutions are respectively 2.5mg/mL, 1.25mg/mL, 0.625mg/mL, 0.313mg/mL, 0.156mg/mL and 0 mg/mL; the rinsing liquid is methanol water solution, and the volume concentration of the rinsing liquid is 50 percent; the eluent is sodium hydroxide solution, and the molar concentration of the eluent is 1 mol/L.
Example 2
This embodiment provides a kit for detecting protein content, which comprises amino black staining solution, rinsing solution, eluent and several sets of standard protein solutions with different concentrations. In addition, the kit may further include a petri dish, a punch, a nitrocellulose membrane, etc. required for the detection process, but is not limited thereto.
Wherein, the amino black staining solution is obtained by adding 20mL of methanol, 5mL of glacial acetic acid and 0.05g of amino black into a container, adding water to a constant volume of 100mL, and uniformly mixing; the plurality of groups of standard protein solutions with different concentrations are standard bovine serum albumin solutions, and the concentrations of the standard protein solutions are respectively 2.5mg/mL, 1.25mg/mL, 0.625mg/mL, 0.313mg/mL, 0.156mg/mL and 0 mg/mL; the rinsing liquid is methanol water solution, and the volume concentration of the rinsing liquid is 40 percent; the eluent is sodium hydroxide solution, and the molar concentration of the eluent is 0.5 mol/L.
Example 3
This embodiment provides a kit for detecting protein content, which comprises amino black staining solution, rinsing solution, eluent and several sets of standard protein solutions with different concentrations. In addition, the kit may further include a petri dish, a punch, a nitrocellulose membrane, etc. required for the detection process, but is not limited thereto.
Wherein, the amino black staining solution is obtained by adding 40mL of methanol, 15mL of glacial acetic acid and 0.15g of amino black into a container, adding water to a constant volume of 100mL, and uniformly mixing; the plurality of groups of standard protein solutions with different concentrations are standard bovine serum albumin solutions, and the concentrations of the standard protein solutions are respectively 2.5mg/mL, 1.25mg/mL, 0.625mg/mL, 0.313mg/mL, 0.156mg/mL and 0 mg/mL; the rinsing liquid is methanol water solution, and the volume concentration of the rinsing liquid is 60 percent; the eluent is sodium hydroxide solution, and the molar concentration of the eluent is 1.5 mol/L.
Example 4
This embodiment provides a kit for detecting protein content, which comprises amino black staining solution, rinsing solution, eluent and several sets of standard protein solutions with different concentrations. In addition, the kit may further include a petri dish, a punch, a nitrocellulose membrane, etc. required for the detection process, but is not limited thereto.
Wherein, the amino black staining solution is obtained by adding 25mL of methanol, 8mL of glacial acetic acid and 0.08g of amino black into a container, adding water to a constant volume of 100mL, and uniformly mixing; the plurality of groups of standard protein solutions with different concentrations are standard bovine serum albumin solutions, and the concentrations of the standard protein solutions are respectively 2.5mg/mL, 1.25mg/mL, 0.625mg/mL, 0.313mg/mL, 0.156mg/mL and 0 mg/mL; the rinsing liquid is methanol water solution, and the volume concentration of the rinsing liquid is 45 percent; the eluent is sodium hydroxide solution, and the molar concentration of the eluent is 0.8 mol/L.
Example 5
This embodiment provides a kit for detecting protein content, which comprises amino black staining solution, rinsing solution, eluent and several sets of standard protein solutions with different concentrations. In addition, the kit may further include a petri dish, a punch, a nitrocellulose membrane, etc. required for the detection process, but is not limited thereto.
Wherein, the amino black staining solution is obtained by adding 35mL of methanol, 12mL of glacial acetic acid and 0.12g of amino black into a container, adding water to a constant volume of 100mL, and uniformly mixing; the plurality of groups of standard protein solutions with different concentrations are standard bovine serum albumin solutions, and the concentrations of the standard protein solutions are respectively 2.5mg/mL, 1.25mg/mL, 0.625mg/mL, 0.313mg/mL, 0.156mg/mL and 0 mg/mL; the rinsing liquid is methanol water solution, and the volume concentration of the rinsing liquid is 55 percent; the eluent is sodium hydroxide solution, and the molar concentration of the eluent is 1.2 mol/L.
Example 6
This embodiment provides a method for detecting protein content, which is performed using the kit provided in embodiment 1, and specifically includes the following steps:
s1, adding 10 mu L of Loading Buffer into 40 mu L of protein sample to be detected, placing the mixture at 95 ℃ for denaturation treatment for 5min, and quickly placing the mixture on ice for cooling. And (5) taking 5 mu L of each sample, vertically dropping the sample on the nitrocellulose membrane, and putting the nitrocellulose membrane in a rinsing solution for rinsing after the liquid is dried. It should be noted that, if the protein sample to be detected has been subjected to Loading Buffer water boiling lysis during collection and extraction, the Loading Buffer may not be added in this step. The method for collecting the protein sample to be detected can comprise the following steps:
(1) cell sample: the main methods include a circulating freeze thawing method, an osmosis method, an ultrasonic method, a detergent method, an enzymatic cracking method and a mechanical homogenizing method. Generally, enzymatic cleavage is used. The method specifically comprises the following steps: centrifuging the obtained cells at 1500rpm for 5min, discarding the supernatant, adding an appropriate volume of RIPA lysate (PMSF before use), performing ice lysis for 1.5h, centrifuging at 4 ℃ and 1500rpm for 10min, and taking the supernatant as the protein sample to be detected.
(2) Tissue sample: cutting the tissue into small pieces, carrying out ultrasonic disruption, and adding RIPA lysate into the disrupted cells for lysis.
(3) A thallus sample: the common method is to add a Loading Buffer after ultrasonic crushing for boiling and cracking, or directly add the Loading Buffer for boiling and cracking.
Alternatively, the cytoplasm (soluble protein) can be collected using Tris-HCl; cytosol (scaffold binding protein) can be collected using Tris-Triton; membrane proteins can be collected using NP-40 or RIPA; the nucleoprotein can be collected by RIPA or a commercially available nucleoprotein extraction kit; mitochondrial proteins can be collected using RIPA or commercially available mitochondrial isolation kits.
S2, after the color of the rinsed protein sample point to be detected disappears, putting the nitrocellulose membrane into a culture dish added with 20mL of amino black staining solution for staining for 8 min.
S3, after the color of the sample point is stable, taking out the nitrocellulose membrane, and rinsing the nitrocellulose membrane in the rinsing liquid for three times, wherein each time is 1 min; and (3) after the surface of the nitrocellulose membrane is clean and the sample point is not faded, airing the nitrocellulose membrane, taking down the blue part at the sample point by using a puncher, putting the blue part into 1mL of eluent for vortex oscillation for 40s, standing the eluent at room temperature for 5min, and oscillating the eluent for 50s again for uniform mixing to obtain the test solution.
S4, taking a 96-well plate, sucking 200 mu L of the test solution, adding into the hole, setting 3 times of repetition, simultaneously using eluent as a control, and measuring absorbance at A650nm to obtain a measured OD value; substituting the measured OD value into the standard curve of the kit provided in the above embodiment 1, so as to calculate the concentration value of the protein in the protein sample to be detected. The method for drawing the standard curve of the kit provided in the above example 1 is as follows: the standard protein solutions with different concentrations in the kit provided in the above example 1 are respectively substituted for the protein sample to be detected, and the detection is performed according to the same method, and then a standard curve can be drawn according to the OD value obtained by the detection and the protein concentration of the corresponding standard protein solution, as shown in fig. 1.
Example 7
This embodiment provides a method for detecting protein content, which is performed using the kit provided in embodiment 4 above, and specifically includes the following steps:
s1, adding 10 mu L of Loading Buffer into 40 mu L of protein sample to be detected, placing the mixture at 93 ℃ for denaturation treatment for 5min, and then quickly placing the mixture on ice for cooling. And (5) taking 5 mu L of each sample, vertically dropping the sample on the nitrocellulose membrane, and putting the nitrocellulose membrane in a rinsing solution for rinsing after the liquid is dried.
S2, after the color of the rinsed protein sample point to be detected disappears, putting the nitrocellulose membrane into a culture dish added with 15mL of amino black staining solution for staining for 5 min.
S3, after the color of the sample point is stable, taking out the nitrocellulose membrane, and rinsing the nitrocellulose membrane in the rinsing liquid for three times, wherein each time is 1 min; and (3) after the surface of the nitrocellulose membrane is clean and the sample point is not faded, airing the nitrocellulose membrane, taking down all the blue part at the sample point by using a puncher, placing the blue part in 1mL of eluent for vortex oscillation for 30s, placing the eluent at room temperature for 5min, and oscillating the eluent for 30s again for uniform mixing to obtain the test solution.
S4, taking a 96-well plate, sucking 200 mu L of the test solution, adding into the hole, setting 3 times of repetition, simultaneously using eluent as a control, and measuring absorbance at A650nm to obtain a measured OD value; substituting the measured OD value into the standard curve of the kit provided in the above embodiment 4, so as to calculate the concentration value of the protein in the protein sample to be detected. The method for drawing the standard curve of the kit provided in the above example 4 is as follows: the standard protein solutions with various concentrations in the kit provided in the above embodiment 4 are respectively substituted for the protein sample to be detected for detection, and then a standard curve can be drawn according to the OD value obtained by detection and the protein concentration of the corresponding standard protein solution.
Example 8
This embodiment provides a method for detecting protein content, which is performed using the kit provided in embodiment 5 above, and specifically includes the following steps:
s1, adding 10 mu L of Loading Buffer into 40 mu L of protein sample to be detected, placing the mixture at 97 ℃ for denaturation treatment for 5min, and quickly placing the mixture on ice for cooling. And (5) taking 5 mu L of each sample, vertically dropping the sample on the nitrocellulose membrane, and putting the nitrocellulose membrane in a rinsing solution for rinsing after the liquid is dried.
S2, after the color of the rinsed protein sample point to be detected disappears, putting the nitrocellulose membrane into a culture dish added with 25mL of amino black staining solution for staining for 10 min.
S3, after the color of the sample point is stable, taking out the nitrocellulose membrane, and rinsing the nitrocellulose membrane in the rinsing liquid for three times, wherein each time is 1 min; and (3) after the surface of the nitrocellulose membrane is clean and the sample point is not faded, airing the nitrocellulose membrane, taking down the blue part at the sample point by using a puncher, putting the blue part into 1.5mL of eluent for vortex oscillation for 60s, standing the eluent at room temperature for 5min, and oscillating the eluent for 60s again for uniform mixing to obtain the test solution.
S4, taking a 96-well plate, sucking 200 mu L of the test solution, adding into the hole, setting 3 times of repetition, simultaneously using eluent as a control, and measuring absorbance at A650nm to obtain a measured OD value; substituting the measured OD value into the standard curve of the kit provided in the above embodiment 5, so as to calculate the concentration value of the protein in the protein sample to be detected. The method for drawing the standard curve of the kit provided in example 5 is as follows: the standard protein solutions with various concentrations in the kit provided in the above embodiment 5 are respectively substituted for the protein sample to be detected for detection, and then a standard curve can be drawn according to the OD value obtained by detection and the protein concentration of the corresponding standard protein solution.
Experimental example:
a bovine serum albumin sample (40. mu.L) of known concentration: 2.5. mu.g/. mu.L was taken, diluted with 10. mu.L of 5 Xloading Buffer, and then the protein concentration was measured in the same manner as in example 6 above, and the measurement OD values and the protein concentration values of the three measurements were repeated three times as shown in Table 1. Wherein, the contrast OD value of the eluent is 0.05, the sample dilution factor is 1.25, and the calculation formula of the protein concentration value is as follows:
protein concentration value (μ g/μ L) = [ (measured OD value-control OD value +0.0027)/0.0405] × sample dilution factor.
TABLE 1
| Number of detections | Measuring OD value | Protein concentration value (μ g/. mu.L) |
| 1 | 0.128 | 2.49 |
| 2 | 0.125 | 2.40 |
| 3 | 0.127 | 2.46 |
As can be seen from table 1 above, the kit and the method for detecting protein content provided by the embodiment of the present invention can accurately and quantitatively detect the protein concentration in a sample, and the accuracy rate can reach more than 96%.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. The amino black staining solution for detecting the protein content is characterized by comprising the following components per liter: 200-400 mL of methanol, 50-150 mL of glacial acetic acid, 0.05-0.15 g of amino black and the balance of water.
2. The amino black staining solution for detecting protein content according to claim 1, wherein each liter of the amino black staining solution comprises the following components: 250-350 mL of methanol, 80-120 mL of glacial acetic acid, 0.08-0.12 g of amino black and the balance of water.
3. A kit for detecting protein content, which comprises the amino black staining solution according to claim 1 or 2.
4. The kit for detecting protein content according to claim 3, wherein the kit further comprises a rinsing solution, an eluent and several sets of standard protein solutions with different concentrations.
5. The kit for detecting protein content according to claim 4, wherein the standard protein solution is a standard bovine serum albumin solution.
6. The kit for detecting protein content according to claim 4, wherein the rinsing solution is a methanol aqueous solution with a volume concentration of 40-60%.
7. The kit for detecting protein content according to claim 4, wherein the eluent is sodium hydroxide solution with a molar concentration of 0.5-1.5 mol/L.
8. A method for detecting protein content, which is carried out by using the kit according to any one of claims 4 to 7, and comprises the following steps:
respectively detecting a plurality of groups of standard protein solutions with different concentrations, and drawing a standard curve;
after carrying out denaturation treatment on a protein sample to be detected, placing the protein sample on a carrier, and rinsing the protein sample by using the rinsing liquid;
after the color of the rinsed protein sample to be detected disappears, placing the carrier in the amino black staining solution for staining;
after the color of the dyed protein sample to be detected is stable, placing the carrier in the rinsing liquid for rinsing; then, taking out the protein sample to be detected on the carrier, and placing the protein sample in the eluent for oscillation treatment to obtain a test solution;
and carrying out absorbance test on the test solution, and calculating to obtain a concentration value of the protein in the protein sample to be tested according to the standard curve.
9. The method for detecting protein content according to claim 8, wherein the temperature of the denaturation treatment in the step is 93-97 ℃.
10. The method for detecting protein content according to claim 8, wherein the carrier is a nitrocellulose membrane.
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