MarvoBlog — Affinity purification

His tag length influences yield and purity on IMAC column           From time to time, we would get questions like: why is 6xHis (6 constitutive histidine residues) used in most recombinant proteins that are constructed for purification by IMAC? how about 4xHis or 10xHis? how does the length of the polyHis tag impact on IMAC purification?           It is true that the most commonly used polyHis tag consists of 6 constitutive histidine residues. Longer or shorter polyHis tags have been used much infrequently, but they have been proven to be effective in purification of various target proteins. The reason for...

IMAC for purification of poly Histinine tag is highly specific, yet inexpensive separation method to obtain highly pure protein. The poly Histinine tag is rather small and has very minimal impact on the conformation and function of the recombinant proteins. However, every protein is different. No one protocol fits all. There are many challenges that scientists have been encountering in purifying their proteins. At Marvelgent, we provide tool tips and tricks based on our knowledge and experience, and hope you will find them helpful in tackling the challenges.   Challenge Two: Refolding of protein from E. coli inclusion bodies  ...

     The purification conditions allowing for the maximal protein yield and purity may vary significantly depending on the characteristics of a given target protein. Finding the best conditions frequently require optimizing the key parameters through small-scale trial and error approaches. 1. Setting up binding conditions for target protein      In most successful purification protocols, the ratio for binding is 1 mL of resins for every 10 – 50 mL of eukaryotic culture supernatant; and 1 mL for 10 ~ 20 mL E.coli lysate.      To set up protocol to purify a new protein, it is recommended to start with the...

The property and performance of each IMAC resin format is determined by the combination of three components of the resin: the matrix support, the chelate ligand that is used to functionalize the matrix surface, and the immobilized metal ion that is in complex with the ligand. Combinations of these three variables (matrix, ligand, and ion) provide a collection of IMAC resins that we can choose from for our specific purification needs. Choosing the appropriate IMAC resin format contributes to the success of obtaining desired amount of pure target His-tagged protein for downstream applications. Read our Tech Note on how to...