Method for preparing saliva protein sample

A salivary protein and sample technology, which is applied in the field of preparation of whole salivary protein, can solve the problems of affecting the separation effect, sample volume error, viscous salivary mucin, etc., achieve high protein recovery rate, increase sample volume, The effect of improving stability

Active Publication Date: 2010-10-27
ZHEJIANG CHINESE MEDICAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

However, due to the viscous nature, rich content (accounting for about 7%-20% of total protein) and large molecular weight of salivary mucin, it will cause experimental errors and affect the repeatability of experiments in the preparation and separation analysis of salivary proteome samples.
For example, the viscous nature of mucin will cause errors in the amount of sample loaded; the large molecular weight of mucin will block the pore size of the electrophoresis gel and affect the separation effect; as a high-abundance protein, mucin will affect other low-abundance proteins in the protein sample. Loading amount of

Method used

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  • Method for preparing saliva protein sample
  • Method for preparing saliva protein sample
  • Method for preparing saliva protein sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1, saliva sample collection

[0016] Six saliva samples were obtained from healthy female graduate students in our laboratory. Aged 24 to 29 years old. The saliva collection time was from 9:00 to 12:00 in the morning, and water was fasted 2 hours before the collection. Sit quietly on a chair after gargling with water, swallow the saliva naturally within the first 5 minutes and start collecting it. After the oral saliva has accumulated to a certain amount, spit it into a 50ml centrifuge tube placed in an ice bath, and collect a total of 6ml of saliva samples ( 3ml for each group), the collection time is 5-10min.

Embodiment 2

[0017] Embodiment 2, salivary protein sample preparation and determination

[0018] Method 1: Centrifuge the sample collected in Example 1 for 15 minutes (4°C, 800×g), absorb the supernatant, add twice the volume of 0.15% dilute acetic acid, mix well, and centrifuge again for 60 minutes (4°C, 14000×g) , absorb the supernatant, put it into a dialysis bag and dialyze with double distilled water for 5h, and then use polyethylene glycol 20000 to concentrate to about 300μl.

[0019] Method 2: Centrifuge the sample collected in Example 1 for 15 minutes (4°C, 800×g), absorb the supernatant, centrifuge again for 60 minutes (4°C, 14000×g), absorb the supernatant, and take the pre-cooled to 4°C Acetone, according to the ratio of 4:1 (acetone: sample), pour into the sample, mix well, store at -20°C for 24h, take out the sample, centrifuge for 60min (4°C, 14000×g), discard the supernatant, see the centrifuge tube There was a white flocculent substance at the bottom, and the precipitate w...

Embodiment 3

[0031] Embodiment 3, the influence of acetic acid volume and concentration on the preparation of salivary protein sample

[0032] 1. The experimental method is as follows:

[0033] (1) Saliva sample collection

[0034]The saliva collection time was from 9:00 to 12:00 in the morning, and water was fasted 2 hours before the collection. Sit quietly on a chair after rinsing your mouth with clean water, swallow the saliva naturally within the first 5 minutes, and start collecting it. After the oral saliva has accumulated to a certain amount, spit it into a 50ml centrifuge tube placed in an ice bath, and collect a total of 12ml of saliva samples ( 100 μl per tube, 6 tubes per group).

[0035] (2) Different volumes of dilute acetic acid precipitation method for salivary protein sample preparation

[0036] The collected samples were centrifuged for 15min (4°C, 800×g), the supernatant was drawn, and 12 times, 8 times, 4 times, 2 times, 1.5 times, 1 times, 0.5 times, 0 times the volu...

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Abstract

The invention discloses a method for preparing a saliva protein sample, and belongs to the technical field of preparation of saliva protein samples. The method is characterized by comprising the following steps of: 1) collecting saliva; 2) centrifuging 800g of collected saliva at 4 DEG C for 15 minutes, and sucking supernate; 3) adding 0.05 to 5 volume percent acetic acid and uniformly mixing; 4) centrifuging 14,000g of the mixed solution at 4 DEG C for 60 minutes, and sucking supernate; 5) putting the supernate in a dialysis bag, and dialyzing by using double distilled water for 5 hours; and 6) condensing to obtain the saliva protein sample. The method has the advantages of high protein recovery rate, short preparation time, safety, nontoxicity, increase of loading quantity of low abundance proteins, clear and easily analyzed maps and the like.

Description

technical field [0001] The invention belongs to the technical field of electrophoresis protein sample preparation, and in particular relates to a preparation method related to whole salivary protein. Background technique [0002] In recent years, the clinical diagnostic value of saliva has gradually been recognized by people. Saliva is obtained in a non-invasive way, without trauma to the body, can be used for large sample screening, and is conducive to longitudinal dynamic observation. Therefore, more and more clinicians apply the strategy of salivary proteomics to the study of the types and functions of salivary proteins under physiological and pathological conditions. However, because saliva contains a large amount of mucin, it will cause difficulties in sample preparation and errors in separation and analysis in proteomics work, which will affect the repeatability of experiments. [0003] Human salivary mucins are covalent complexes of proteins and carbohydrates, which...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N1/36G01N1/40G01N33/68G01N27/447
Inventor 范永升温成平丁慧登卢德赵曲建全谢志军
Owner ZHEJIANG CHINESE MEDICAL UNIVERSITY
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