How does NI-NTA purification work?
The Ni-NTA Purification System is designed for purification of 6xHis-tagged recombinant proteins expressed in bacteria, insect, and mammalian cells. The system is designed around the high affinity and selectivity of Ni-NTA Agarose for recombinant fusion proteins that are tagged with six tandem histidine residues.
What binds to Ni-NTA during protein purification?
NTA binds metal ions tightly, allowing use of stringent washes. Histidine residues on the tag, connected via a short linker to the C- or N-terminus of the protein, bind to the Ni-ions. The protein can be eluted by competitive displacement with imidazole.
What is a resin purification?
Antibody purification using thiophilic resins is a simple, powerful, and economical alternative to Protein A, the most widely used method of immunoglobulin purification. It is an economical alternative to Protein A for purifying antibodies from whole serum and tissue culture.
What is Ni-NTA affinity chromatography?
Ni-NTA Agarose is a nickel-charged affinity resin that can be used to purify recombinant proteins containing a polyhistidine (6xHis) sequence. Ni-NTA uses the chelating ligand nitrilotriacetic acid (NTA) coupled to a cross-linked 6% agarose resin that is suitable for use in batch and gravity flow applications.
How much resin do you use for protein purification?
To set up protocol to purify a new protein, it is recommended to start with the ratio of 1 mL resin for every 20 mL of culture supernatant or lysate. It also helps to estimate the amount of target protein in the sample and adjust it to approximately 0.5 mg/mL.
What pH range is recommended by Ni-NTA resin manufactures for purification?
Things to remember are: use at least 50 to 100 mM Ni-NTA buffer (TRIS, or HEPES, or phosphate, etc…) at a minimum pH of 7.6 and a maximum pH of 9 (around pH 8 is best unless your protein has a pI of 8, which means your protein would be unstable at pH = 8).
How do you affinity purify antibodies?
Antigen-specific affinity—affinity purification of only those antibodies in a sample that bind to a particular antigen molecule through their specific antigen-binding domains. This purifies all antibodies that bind the antigen without regard to antibody class or isotype.
How does protein A purification work?
A fundamental step in studying individual proteins is purification of the protein of interest. There are four basic steps of protein purification: 1) cell lysis, 2) protein binding to a matrix, 3) washing and 4) elution.
What are Protein A resins?
GenScript Protein A Resin is an affinity chromatography medium designed for easy, one-step purification of classes, subclasses and fragments of immunoglobulins from biological fluids and from cell culture media. Protein A Resin can also be used for immunoprecipitation of proteins, protein complexes or antigens.
What is plant resin?
In polymer chemistry and materials science, resin is a solid or highly viscous substance of plant or synthetic origin that is typically convertible into polymers. Plants secrete resins for their protective benefits in response to injury. The resin protects the plant from insects and pathogens.
What is the Ni-NTA purification system?
The Ni-NTA Purification System is designed for the purification of recombinant proteins that contain a polyhistidine (6xHis) sequence. The kit utilizes Ni-NTA nickel-chelating resin and is supplied with native and denaturing buffers for efficient purification of recombinant proteins under different conditions. For Research Use Only.
What is Ni NTA resin used for?
Ni-NTA Resin. The Ni-NTA Resin is specifically designed for the purification of recombinant proteins fused to the 6 x histidine (6XHis) tag expressed in bacteria, insects, and mammalian cells. The resin is high affinity and selectivity for recombinant fusion proteins that are tagged with six tandem histidine residues.
What is Ni-NTA affinity purification?
Purification of His-Tagged Proteins Ni-NTA affinity purification of His-tagged proteins is a bind-wash-elute procedure that can be performed under native or denaturing conditions. Here, protocols for purification of His-tagged proteins under native, as well as under denaturing conditions, are given.
How do I use hispur Ni-NTA resin?
The HisPur Ni-NTA Resin allows for purification strategy customization. Purification conditions can be scaled as needed. The procedure may be performed at room temperature or at 4°C. Add an appropriate amount of Ni-NTA resin to a tube. Centrifuge tube for 2 minutes at 700 × g and carefully remove and discard the supernatant.