Novel protein molecular weight standard and preparation method thereof
A molecular weight standard, protein technology, applied in the field of bioengineering, can solve the problems of inability to develop color, inability to correctly determine the target protein, etc.
Inactive Publication Date: 2013-05-15
HANGZHOU SONGHUA BIOLOGICAL TECH
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Problems solved by technology
In the Western blotting experiment, because the conventional protein molecular weight standard cannot react with the antibody of the target protein and the corresponding secondary antibody, the color cannot be developed, especially the fluorescence cannot be developed on the film, resulting in the inability to pass the protein molecular weight standard. Auxiliary and correctly judge the correct position of the target protein on the film
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[0010] The present invention will be described in further detail below in conjunction with specific embodiments and accompanying drawings, and the experimental examples are intended to specifically illustrate the present invention by way of example. Reagents, concentrations of reagents, values of temperature and other variables, and selection of vectors and hosts are merely illustrative of the utility of the invention and are not intended to be limiting of the invention. The following description is not intended to limit the scope of the invention.
[0011] 1. Optimizing the nucleotide sequence encoding the SPA protein
[0012] In order to increase the expression of the SPA protein, on the premise that the amino acid sequence of the SPA protein remains unchanged, the nucleotide sequence encoding the SPA protein is optimized using Escherichia coli preferred codons. The optimized nucleotide sequence is SEQ ID NO in the sequence table :1.
[0013] 2. Construction of SPA multi...
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The invention belongs to the technical field of biological engineering, and discloses a novel protein molecular weight standard and a preparation method thereof. The invention adopts the concrete method that recombinant protein which is obtained by the tandem expression of different numbers of SPA (Staphylococcal protein A) is used as the protein molecular weight standard. The invention also discloses a method for applying the novel protein molecular weight standard so as to cause the novel protein molecular weight standard to be developed on a film by utilizing the capability of being combined with the Fc section of various mammal IgG of SPA. The invention also discloses a method for optimizing an SPA nucleotide sequence by genetic engineering, and an optimized nucleotide sequence. The invention further discloses a method for conducting tandem expression and purification on various SPAs by utilizing BsaI restriction sites.
Description
technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a novel protein molecular weight standard for simultaneously displaying proteins with different molecular weights in western blotting experiments. The protein molecular weight standard is composed of a series of recombinant proteins with different numbers of SPA connected in series. Background technique [0002] Western Blot is widely used in immunology. It uses polyacrylamide gel electrophoresis to separate protein samples and transfers them to solid phase supports (such as nitrocellulose membranes). The protein is adsorbed in the form of valence bond, and the type of protein separated by electrophoresis and its biological activity can be kept unchanged. Use the protein on the solid phase carrier as the antigen, incubate with the corresponding antibody, then incubate with the enzyme-labeled secondary antibody, develop color or generate fluorescence with the su...
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IPC IPC(8): G01N33/68C12N15/70C12R1/19
Inventor 余铭恩冯俊涛吴琼杉李晓照敖翔余卫
Owner HANGZHOU SONGHUA BIOLOGICAL TECH