CN103149067A - Method and reagent for extracting fecal protein - Google Patents
Method and reagent for extracting fecal protein Download PDFInfo
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- CN103149067A CN103149067A CN2013100622768A CN201310062276A CN103149067A CN 103149067 A CN103149067 A CN 103149067A CN 2013100622768 A CN2013100622768 A CN 2013100622768A CN 201310062276 A CN201310062276 A CN 201310062276A CN 103149067 A CN103149067 A CN 103149067A
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Abstract
The invention relates to a method and a reagent for extracting fecal protein, and belongs to the extraction and purification field of the protein. A kit used for extracting and purifying the fecal protein comprises an excrement sampler, a micropore plate and an extracting solution. The method for extracting the fecal protein can be used for effectively extracting the fecal protein for detection by the combining action of mechanical treatment and the extracting solution, and has the characteristics of being simple and convenient, high in yield, small in sample capacity, and capable of reducing the pollution and the like, thus being used for pretreatment of detection on fecal protein sample enzyme-linked immunoassay (ELISA) and the like.
Description
Technical field
The present invention relates to Protein Extraction purifying field.Particularly, the present invention relates to method and the reagent of Protein Extraction purifying in a kind of ight soil.
Background technology
During enterogastric diseases, often contain the inflammatory cell (comprising leucocyte, lymphocyte) of more amount, the gastrointestinal epithelial cell that comes off and intestines and stomach microorganism and metabolic product thereof etc. in ight soil.To the analysis of some specific ight soil albumen, can the auxiliary diagnosis enterogastric diseases.
Existing as ight soil lactoferrin enzyme-linked immunologic detecting kit and calprotectin enzyme-linked immunologic detecting kit, all need to carry out protein extraction and purifying to fecal sample before detection.Bibliographical information is arranged, and the ight soil protein extraction can add a certain proportion of urea with a certain proportion of phosphate and extract purifying.One aspect of the present invention has been optimized the ratio of phosphate and urea on the basis of literature method, greatly improved the yield of ight soil albumen; On the other hand, be equipped with a kind of easy dejecta sampling device and centrifuge tube in kit, make the ight soil weighing, extract more convenient, more accurate.
Summary of the invention
The object of the present invention is to provide that a kind of yield is high, easy, ight soil protein extraction purification kit accurately, specifically, this kit has comprised sampler, centrifuge tube, extract.
Wherein sampler is the ring texture as shown in the figure of being made by metal or plastics.
Described centrifuge tube is that capacity is company's lid centrifuge tube of 15ml.
Described microwell plate is ELISA Plate or the microwell plate of every pore capacities 300ul.
Described extract phosphate-containing and urea more preferably extract the protein in ight soil.
Another aspect of the present invention provides a kind of extraction purifying ight soil method of protein, comprises step:
(1) sample sampling weighing:
(2) carry out extraction and purification with the arbitrary described kit of claim 1-4, load weighted fecal sample is added in centrifuge tube, add 2 times of extracts after dilution, abundant mixing, shook 20 minutes, get the 1ml crude extract to the 1.5mlEP pipe, 2000rpm/ divided centrifugal 20 minutes, drew supernatant in microwell plate.
(3) analyzing proteins yield.
The detection principle of kit of the present invention is:
Extract contains can make protein-denatured urea, makes cell rupture by mechanical oscillation, and the protein dissolution in ight soil then removes impurity in ight soil with centrifugal method in solution, just can obtain containing the supernatant of the protein of higher concentration.
Description of drawings:
Sampler structure figure.
It is in order to understand better the present invention that following embodiment is provided, rather than content of the present invention and protection domain are consisted of any restriction.
The impact of embodiment 1 extract principal ingredient concentration on ight soil protein extraction yield
1.1 sample sampling weighing: the described sampler of claim 1-4 and centrifuge tube are carried out weighing, again carry out weighing after getting the 50-200mg fecal sample and be transferred to centrifuge tube with sampler.Two numbers subtract each other the weight that obtains fecal sample.
1.2 phosphate and urea are formulated as many parts of extracts (table 1) by variable concentrations with chessboard method, and 2 times of diluted for use.
1.3 ight soil protein extraction: (V is extracting liquid volume, the ul of unit by V: W=50; W is sample weight, and unit is mg) ratio add extract in centrifuge tube, fully mixing, shake 20 minutes, got the 1ml crude extract to the 1.5mlEP pipe, 3000rpm/ divided centrifugal 15 minutes, the absorption supernatant.Repeat this step to the extract of all proportionings all with appropriate sample effect and obtain supernatant.With PBS, supernatant is distinguished 2 times of dilutions.
1.4 analyzing proteins yield: add 1 volume Cu reagent (50: 1) with 50 volume BCA reagent and be mixed with working fluid, add 20ul supernatant/standard items and 200ul working fluid in 96 orifice plates, hatched 15 minutes for 37 ℃.At the 562nm reading, gained supernatant OD value (the high expression of OD value protein concentration is high) is as table 1 with microplate reader.
Table 1 extract concentration primary dcreening operation (OD value)
By typical curve (y=1567.3x-8.1326, R
2=0.9976) get protein concentration:
Table 2 extract concentration primary dcreening operation (protein concentration, ng/ml)
1.5 the result by table 2 is done suitable adjustment to urea and phosphatic concentration, repeats 1.1-1.4 step in example 1, acquired results sees Table 3.
Table 3 extract concentration adjustment (OD value)
By typical curve (y=1543.1x-1.8418, R
2=0.9977) protein concentration that converts to get:
Table 4 extract on the impact of ight soil protein extraction yield (protein concentration, ng/ml)
In definite extract, phosphate and the applicable concentration of urea are respectively thus: 0.1M-1M and 1-9M.
The impact on ight soil protein extraction yield of embodiment 2Tris and pH value
2.1 add respectively 0.05M, 0.1M in phosphate-urea liquid (phosphate 0.3M, urea 3M), 0.15M, 0.2MTris make different extracts, press the 1.4 same samples of described mensuration gained ight soil protein content after different extracts are processed in example 1, the results are shown in Table 5.
The impact (OD value) of table 5 variable concentrations Tris on the ight soil protein extraction
Can get thus protein concentration (typical curve y=2075.1x-128.01, R
2=0.9921)
Table 6 variable concentrations Tris on the impact of ight soil protein extraction (protein concentration, ng/ml)
According to table 6 result, choosing 0.05M-0.2M Tris is optimum concentration.
2.2pH the impact of value on ight soil protein extraction yield: adjust extract (phosphate 0.3M with NaOH, urea 3M, Tris0.15M) pH value is 7.5,7.8,8.0,8.2,8.5,9.0, press the 1.4 same samples of described mensuration gained ight soil protein content after different extracts are processed in example 1, the results are shown in Table 6.
The impact (OD value) of the different pH values of table 7 on the ight soil protein extraction
Can get (typical curve y=1855.6x+25.482, R
2=0.9915):
The different pH values of table 8 on the impact of ight soil protein extraction (protein concentration, ng/ml)
According to table 8 result, choosing pH8.5 is optimum pH.
Conclusion phosphate and urea concentration are respectively 0.3M, and 3M and the pH8.5 extract that contains 0.15M Tris are the suitableeest extract.
Claims (9)
1. ight soil protein extraction kit, comprising: dejecta sampling device, 15ml centrifuge tube, extract.
2. kit according to claim 1, is characterized in that, described dejecta sampling device is by metal or plastic products as graph structure.
3. extract according to claim 1, is characterized in that, described extract contains 0.1M-1M phosphate and 1M-9M urea.
4. according to claim 1 or 3 described extracts, is characterized in that, described extract contains 0.3M phosphate and 3.0M urea.
5. extract according to claim 1, is characterized in that, described extract contains 0.05M-2.0M Tris.
6. extract according to claim 1 or 5, is characterized in that, described extract contains 0.15M Tris.
7. extract according to claim 1, is characterized in that, described extract pH is 8.0-9.0.
8. according to claim 1 or 7 described extracts, is characterized in that, described extract pH is 8.5.
9. the method for protein extraction purifying in a fecal sample comprises step:
(1) sample sampling weighing;
(2) extract purifying with the arbitrary described kit of claim 1-4, take out fecal sample with sampler, use electronic balance weighing, put into the 15ml centrifuge tube together with sampler; With 2 times of extract dilutions, the treating fluid after diluting in the ratio of V: W=50 adds in centrifuge tube, and fully mixing, shook 20 minutes, gets the 1ml crude extract and manage to 1.5mlEP, and 2000 left the heart 20 minutes, got supernatant, namely extracted ight soil albumen;
(3) analyzing proteins yield.
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| CN2013100622768A CN103149067A (en) | 2013-02-28 | 2013-02-28 | Method and reagent for extracting fecal protein |
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| CN2013100622768A CN103149067A (en) | 2013-02-28 | 2013-02-28 | Method and reagent for extracting fecal protein |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110511263A (en) * | 2019-09-03 | 2019-11-29 | 北京合力清源科技有限公司 | A kind of protein extracted from fresh chicken manure and its extracting method |
| CN117907608A (en) * | 2024-03-19 | 2024-04-19 | 苏州和锐生物科技有限公司 | Claudin2 protein detection method and related products and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110511263A (en) * | 2019-09-03 | 2019-11-29 | 北京合力清源科技有限公司 | A kind of protein extracted from fresh chicken manure and its extracting method |
| CN117907608A (en) * | 2024-03-19 | 2024-04-19 | 苏州和锐生物科技有限公司 | Claudin2 protein detection method and related products and application thereof |
| CN117907608B (en) * | 2024-03-19 | 2024-05-24 | 苏州和锐生物科技有限公司 | Claudin2 protein detection method and related products and application thereof |
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Application publication date: 20130612 |