The HAT (histidine affinity tag) Protein Expression and Purification System provides a convenient and efficient way to express and purify proteins. HAT vectors encode a novel polyhistidine epitope tag that allows proteins expressed in bacteria to be purified at neutral or physiological pH and under native or denaturing conditions.
The HAT (histidine affinity tag) Protein Expression and Purification System provides a convenient and efficient way to express and purify proteins. HAT vectors encode a novel polyhistidine epitope tag that allows proteins expressed in bacteria to be purified at neutral or physiological pH and under native or denaturing conditions.
The HAT system
The HAT (histidine affinity tag) epitope is a naturally-occurring sequence of non-adjacent histidine residues that has a lower overall charge than tags with consecutive His residues.
HAT-protein fusions exhibit solubility that more closely resembles that of wild-type proteins, while still possessing strong affinity for immobilized metal ions. Using the HAT system to purify proteins offers two major advantages:
- The protein is soluble, therefore it does not form aggregates or reside within inclusion bodies.
- The protein can be eluted under mild conditions, such as neutral pH or low imidazole concentration.
Proteins expressed from pHAT vectors are ideal for purification with TALON resin using batch or gravity-flow protocols, or with TALON Superflow Resin using medium pressure FPLC. The cobalt metal ion in the TALON resin has a high affinity for HAT and other polyhistidine-tagged proteins, and a very low affinity for other proteins. The exceptional selectivity of TALON resin reduces background and requires fewer washes with milder conditions that preserve protein integrity.
pHAT vectors
The HAT system includes three pHAT vectors—pHAT10/11/12, which are the same construct but have multiple cloning sites (MCS) in each of the three reading frames. An enterokinase (EK) cleavage site has been incorporated into each of the vectors, allowing removal of the HAT sequence from the purified protein. Restriction sites are present to allow the HAT sequence to be excised, with or without the EK site, for cloning into other vectors.
The pHAT20 Vector, available separately, provides all the advantages of the pHAT10/11/12 vectors along with the convenience of additional cloning sites, making it easier to clone your cDNA in-frame for fusion to the HAT sequence.
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抗原是指一种能刺激人或动物机体产生抗体或致敏淋巴细胞,并能与这些产物在体内或体外发生特异性反应的物质。抗原的基本能力是免疫原性和反应原性。免疫原性又称为抗原性,是指能够刺激机体形成特异抗体或致敏淋巴细胞的能力。反应原性是指能与由它刺激所产生的抗体或致敏淋巴细胞发生特异性反应。具备免疫原性和反应原性两种能力的物质称为完全抗原,如病原体、异种动物血清等。只具有反应原性而没有免疫原性的物质,称为半抗原,如青霉素、磺胺等。半抗原没有免疫原性,不会引起免疫反应。但在某些特殊情况下,如果半抗原和大分子蛋白质结合以后,就获得了免疫原性而变成完全抗原。
一个制造一种专一抗体的浆细胞进行培养,就可得到由单细胞经分裂增殖而形成细胞群,即单克隆。单克隆细胞将合成针对一种抗原决定簇的抗体,称为单克隆抗体。
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