Imac Purification
Imac Purification - Related Videos
10:43
Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein
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Cited by 5 •
2010
In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.
07:51
Chromatography-based Biomolecule Purification Methods
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2023
In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by biochemists are size-exclusion chromatography and affinity chromatography. In size-exclusion chromatography, a column packed with porous beads separates components of a mixture based on size. On the other hand, affinity chromatography allows for a more specific separation of biomolecules by using a column that is composed of stationary phase,...
08:16
Plasmid Purification
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2023
Plasmid purification is a technique used to isolate and purify plasmid DNA from genomic DNA, proteins, ribosomes, and the bacterial cell wall. A plasmid is a small, circular, double-stranded DNA that is used as a carrier of specific DNA molecules. When introduced into a host organism via transformation, a plasmid will be replicated, creating numerous copies of the DNA fragment under study. In this video, a step-by-step generalized procedure is described for how to perform plasmid purification.
06:30
Gel Purification
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2023
Gel purification is used to recover DNA fragments after electrophoretic separation. DNA recovery from an agarose gel includes three basic steps: binding, washing and eluting from a silica column. DNA is believed to bind to silica in the presence of high salt via a salt bridge. Following binding, DNA is washed of impurities and eluted under low salt conditions disrupting this interaction. This video goes through a step-by-step, generalized procedure for cutting out a band from the gel, gel...
11:22
GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins
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Cited by 18 •
2013
In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.
FAQs
The polyhistidine tag creates the principal recognition point for capture. Histidine residues bind metal ions that are coordinated to the chromatography matrix, allowing the tagged recombinant protein to remain associated with the solid phase while many other lysate proteins do not. This molecular interaction gives the method its selectivity during separation.
Selectivity depends on the contrast between tagged-protein binding and nonspecific retention by other lysate components. Washing is therefore an important separation stage rather than a purely preparative rinse: it removes proteins that do not remain specifically associated with the immobilized metal sites. Effective washing improves enrichment of the recombinant target before elution.
Elution changes the conditions that maintain target binding. Imidazole provides one release condition, while an altered buffer can also release the protein from the immobilized metal sites. The resulting eluate contains the recovered recombinant protein and can support downstream analysis of its structure, function, interactions, or enzymatic activity.
A typical workflow begins with a complex cell lysate, applies it to a chromatography matrix carrying coordinated nickel or cobalt ions, and allows the tagged protein to bind. The matrix is then washed to remove nonspecific proteins, followed by elution with imidazole or an altered buffer. These stages separate capture, cleanup, and recovery.
Complex cell lysates contain many proteins, yet a recombinant target with a polyhistidine tag can be retained by the metal-loaded matrix while nonspecific proteins are removed during washing. This pairing of selective capture and cleanup makes the method useful for isolating a target from biologically crowded starting material.
The purified fraction supports downstream studies that require an enriched recombinant protein, including investigations of protein structure, protein function, protein interactions, and enzymatic activity. In biology, IMAC purification therefore serves as a preparation step for experiments that examine what a protein looks like, does, interacts with, or catalyzes.