Protein purification equipment steps about protein purification
Protein purification is widely used in production, protein purification equipment steps for protein purification:
1. To know the coding DNA sequence of the purified protein, consult and related research literature, that is, the gene is highly expressed in those cell functions or enterprise organizational structure, improve the primers for designing the gene, and extract the ARF of the target DNA. It is called the acquisition of different target gene fragments.
2. Protein purification equipment Steps for protein purification, construction of vectors: According to the obtained gene characteristics in prokaryotic or eukaryotic expression vectors, the problem of this step is to construct plasmids, genes of interest, and expression systems. Prokaryotic expression time is short, low cost, and large-scale expression is a priority; some genes are not expressed in E. coli, problems may arise due to codon optimization, and consideration of better activity of purified proteins, there are also many researchers choose Expressed in Pichia pastoris. At this step, codon optimization is very important for successful expression.
3. After the vector is constructed, it is time to express the recombinant protein. The process involves how much inducer to use, the problem of constantly getting the most used protein during working hours, and optimizing the design and expression conditions. Under the induction conditions of the above-mentioned different amounts of inducers, take the supernatant of the bacteria or electrophoresis of the broken cells to see if the protein with the molecular weight of the research object can be obtained. It is a method to determine protein purification based on the hydrophilic/hydrophobic and charged properties of the protein itself. General procedure: Centrifuge to take the phase where the protein is located.
4. For secreted protein expression, supernatant, ultrafiltration, dialysis bag without ultrafiltration device is used instead, and ion exchange column chromatography is used. Basically, this step can purify 95% of the protein, and the presence of heterozygotes will not be seen in electrophoresis identification. Protein purification equipment Regarding the steps of protein purification, if the purity of the expressed protein cannot meet the requirements, when constructing the expression vector, a his tag can be added to the orf end of the expressed protein. When it is uncertain whether the fusion protein is expressed, use an anti-his antibody as a wb analysis Whether the target protein is expressed or not is a commonly used protein expression method. After the purified protein still contains part of the inorganic salts, the protein can be freeze-dried by vacuum freeze-drying machine.
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