Western blot detection method for phosphoprotein

A phosphorylated protein and phosphorylated protein technology, which is applied in biological testing, material inspection products, etc., can solve the problem of low stoichiometric value of phosphorylated protein phosphorylation sites, slow development of phosphorylated proteomics research, and few analytical and detection methods and other problems, to achieve the effect of low instrument requirements, good effect and good stability

Inactive Publication Date: 2018-07-03
XI AN JIAOTONG UNIV +1
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  • Application Information

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Problems solved by technology

However, because the content of phosphorylated proteins in vivo and the stoichiometry of phosphorylated sites are often very low, large-scale analysis of phosphorylated proteins is technically difficult.
At present, the research on phosphoproteomics develops slowly, and there are few related analysis and detection methods

Method used

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  • Western blot detection method for phosphoprotein
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The phosphorylated protein of myosin light chain (MLC) with a molecular weight of 18kD is proposed to be used as the research object, and the effects of different transfer methods and different antibody dilution ratios on the protein and phosphorylated expression detection are compared.

[0023] Follow the steps below:

[0024] S1. Sample preparation and gel electrophoresis: Add 6 times SDS loading buffer to the phosphorylated protein sample at a ratio of 5:1, mix the sample evenly, and boil it at 100°C for 5 minutes in a dry-type thermostat to make the amino acid side chain of the protein Fully combined with SDS. The denaturation efficiency of DTT and SDS proteins can be improved at 100°C; add 1 times the electrophoresis buffer to the electrophoresis device, add the pre-stained indicator and phosphorylated protein samples to the sample wells of the gel, and set a constant voltage of 80V when starting electrophoresis. When the indicator band runs into the separating ge...

Embodiment 2

[0033] The phosphorylated myosin light chain was extracted from mouse tracheal smooth muscle cells. The amount of protein loaded was 60 μg. The antibody was p-MLC (ab2480). For the wet transfer method, the operation was performed according to the steps described in Example 1.

[0034] see figure 2 The comparison chart of the detection results of p-MLC protein expression with different antibody dilution ratios shown shows that: when the antibody dilution ratio is 1:3000, non-specific bands appear with a molecular weight of 25kD ( figure 2 A); When the antibody dilution ratio is reduced to 1:5000, there are no non-specific bands, and the protein signal effect is better, and the average gray value is 59563 ( figure 2 B) When the antibody dilution ratio is 1:10000, there is also no non-specific band, but the band appears diffuse and the background is high, and the average gray value is 50387 ( figure 2 C).

[0035] In the immunoblotting detection of phosphorylated proteins,...

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Abstract

The invention discloses a western blot detection method for phosphoprotein. The western blot detection method for the phosphoprotein comprises the following steps: S1, preparing a sample, carrying outgel electrophoresis, namely adding 6 times SDS sample loading buffer solution in a ratio of 5:1 into the phosphoprotein sample, uniformly mixing, and carrying out electrophoresis at a voltage of 80 V; S2, transferring a membrane, namely connecting a gel surface with a negative pole, connecting a PVDF membrane with a positive pole, and carrying out transfer printing for 1 hour at a constant voltage of 130 V; S3, sealing, namely putting the PVDF membrane into PBST containing 5% of BSA, and hatching for 20 minutes in a shaking table and at room temperature; S4, adding primary antibodies, namelyadding the primary antibodies, hatching for 2 hours in the shaking table and at the temperature of 24 DEG C, and rinsing for four times with PBST, wherein the rinsing is carried out once every 5 minutes; S5, adding secondary antibodies, namely adding anti-rabbit secondary antibodies labelled by horse radish peroxidase (HRP), hatching for 1 hour in the shaking table and at room temperature, and rinsing for four times with PBST, wherein the rinsing is carried out once every 5 minutes; and S6, developing, namely uniformly dropwise adding a developing reagent on the PVDF membrane, and carrying outgel developing. The western blot detection method for the phosphoprotein has the advantages that a wet membrane transferring manner is adopted during western blot detection of the phosphoprotein, anda protein signal is continuous and high in strength; and no non-specific band exists when dilution ratio of the primary antibodies is 1:5000, and the protein signal is better in effect.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular, the invention relates to an immunoblotting detection method of phosphorylated protein. Background technique [0002] At present, the incidence of malignant tumors is getting higher and higher. Clinically, CT scans are mainly used for disease screening, which has a blind spot for early screening and diagnosis of small tumor tissues. It is very important to study the molecular mechanism of the occurrence and development of malignant tumors and carry out accurate detection at the molecular level for timely prediction and screening of early disease. Phosphorylation modification of protein is one of the most important covalent modification methods in organisms, and it participates in the regulation of various life activities of cells, including cell proliferation, development and differentiation, cytoskeleton provisions, cell apoptosis, tumorigenesis, etc. Such as tyrosine...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 刘华东祁红斌王震刘健康
Owner XI AN JIAOTONG UNIV
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