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
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[0021] Example one
[0022] It is proposed to use the phosphorylated protein of myosin light chain (MLC) with a molecular weight of 18kD as the research object, and compare the effects of different transfer methods and different antibody dilution ratios on the protein and phosphorylation expression detection effect.
[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 place it in a dry thermostat at 100°C and boil for 5 minutes to make the protein amino acid side chain Fully integrated with SDS. The denaturation efficiency of DTT and SDS proteins can be improved at 100°C; add 1x electrophoresis buffer to the electrophoresis device, add the pre-staining indicator and phosphorylated protein sample to the spotting wells of the gel, and set a constant voltage of 80V when starting electrophoresis. When the indicator strip runs into the se...
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[0032] Example two
[0033] Use mouse tracheal smooth muscle cells to extract phosphorylated myosin light chain. The amount of protein loaded is 60μg. The antibody is p-MLC (ab2480). The primary antibody dilution ratios are 1:3000, 1:5000, 1:10000, respectively. For the wet transfer method, follow the steps described in Example 1.
[0034] See figure 2 The comparison chart of the detection results of different antibody dilution ratios to p-MLC protein expression showed 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 is no non-specific band, and the protein signal effect is better, the average gray value is 59563 ( figure 2 B); When the antibody dilution ratio is 1:10000, there are also no non-specific bands, but the bands are diffuse and the background is high, and the average gray value is 50387 ( figure 2 C).
[0035] The present invention a...
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