Protein Purification Animation his tag protein purification

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Protein Purification Animation - his tag protein purification - This animation lecture explains about the histidine tag mediated protein purification. Widely used quick procedure for producing and purifying a protein involved the addition of a peptide tag to the protein of interest in this procedure a segment of DNA encoding the protein of interest will be inserted into a plasmid with a number of key features plasmid contains of replication origin which allows the plasmid to replicate in a host E. coli cells it also contains a gene that confers resistance to the Arab IRA Is someone allowing selection for cells that carry the plasmid or multiple cloning site provides a number of sites for restriction enzymes to cut the plasmid Williams on PDU to digests the plasmid for example it produces blood ends of the DNA of the DNA with the protein coding region is also blended it can be migrated into this plasmid in this example just upstream of the inserted DNA are three pieces that remain from the multiple cloning site CAG code for the amino acid glutamine maxed six hits to redeem code to call the peptide tag upstream are two more codons one of which is a translation start site ATG which calls for methionine are brighter side is derived from the lack of: control when the tag protein is expressed in the cell repressor proteins bind to go creating an obstruction for RNA polymerase one inducer is added to the culture medium the end-user binds to the repressor proteins and allows RNA polymerase to pass through and transcribe the DNA into RNA another important region in the engineered plasmid is a ribosome binding site ribosome binds to the RNA of this site before locating the first AUG start codon and translating the RNA into protein ribosome continues translation until reaching a stop codon when cells transformed with this plasmid reduced to express the tag protein tag protein becomes bonded in the cell constituted up to 30% of the cellular protein with many such cells growing in culture large amount of the tag protein can be produced the next step is to harvest the cells break them open and prepare a crude cell extract a column consisting of all those beads attached to little add-ons can separate the tag proteins from the rest of the cellular material within the column history in tags of the proteins have affinity for and bind to the nickel bearing all the beads are proteins that like the tags do not stick to let go and can be washed out of the car verse the history intact proteins are separated from the others by a process generally called affinity purification returned protein is eluded from the column a low pH solution causes the history residues to become protein needed and unable to bind to the neck or on the beads alternatively a solution with a molecule called imidazole complete with his team for NATO binding in each case the tag proteins are released from the car after the tag protein eludes from the column an enzyme is added to remove the tag from the amino terminal end of the protein another enzyme is added at the same time to modify the glutamine residue at the end of the tag this modification prevents the digestive of the protein of interest the enzymes used in the system have been engineered with his study in tags of the role the tags allow you enzymes to be retained on a nickel-based, while a protein of interest washes through finally another enzyme removes the modified glutamine amino acid this enzyme can be removed later from the mixture by using its history in tag and the nickel-based, to separate the enzyme from the protein of interest the steps involved in producing and purifying the protein of interest can be completed relatively quickly in the period of a week. • Thank You for watching our videos from Biology Animation Videos channel. This channel is created to compile animated biology lectures and videos from different animation sources. None of these videos are created by us. we just organize them and place them in YouTube for your understanding so If you want to know details about these animation please see the credit section for knowing the original content developer and please convey privilege and gratitude to them. Thank You. • Title - • Protein Purification Animation - his tag protein purification • Our website- • https://www.biologyanimationvideos.we...

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