I want to purify His-tagged proteins from them using Ni-NTA column chromatography, so that recombinant tagged proteins remain in their active confirmations.
There are many lysis buffers that you can try but all will depend on the nature of your protein. Just keep in mind that you have to avoid the EDTA and DTT or BME (though lower concentration is permissible) in your buffer during Ni-NTA purification. Secondly, you must consider the pH of your lysis buffer for binding on the beads. Below 6 pH is generally avoided (reason is the protonation state of His). In general you can use 50 mM Tris-Cl (pH7.5 or 8), 200-300 mM NaCl. But you may need to modify the condition as per the behavior of your protein. you can download the protein purification handbook available at qiagen web site. For general consideration follow link given below
http://www.qiagen.com/in/resources/resourcedetail?id=dc89c299-75d3-4120-adf0-35251f16a7af&lang=en
There are many lysis buffers that you can try but all will depend on the nature of your protein. Just keep in mind that you have to avoid the EDTA and DTT or BME (though lower concentration is permissible) in your buffer during Ni-NTA purification. Secondly, you must consider the pH of your lysis buffer for binding on the beads. Below 6 pH is generally avoided (reason is the protonation state of His). In general you can use 50 mM Tris-Cl (pH7.5 or 8), 200-300 mM NaCl. But you may need to modify the condition as per the behavior of your protein. you can download the protein purification handbook available at qiagen web site. For general consideration follow link given below
http://www.qiagen.com/in/resources/resourcedetail?id=dc89c299-75d3-4120-adf0-35251f16a7af&lang=en
When lysing E.coli it is useful to add lysozyme (1 mg/ml) and DNase (eg Benzonase, 0.1 mg/ml) to enhance lysis and reduce the stickiness of the lysate. Also you should consider adding proteinase inhibitors to prevent degradation of your protein by endogenous proteases. I tend to use 500 mM NaCl, 30 mM imidazole, 50 M HEPES pH 8.0 for lysis and as the wash buffer for the NiNTA.
there are different kind of lysis buffer. I always use the lysozyme and the DNase (whit the addiction of MgCl2). I tend to use 500 mM NaCl, 5 / 20 mM imidazole, 50 M Tris-HCl pH 8.0. The concentration of imidazole is dependent on the fusion tag used (for my proteins!!).
Alot of it depends on your target protein. I find you can pretty much use any standard buffer system you like (I have used Tris, TAPS, PO4, Hepes) at around 6-8pH and 25-100mM. NaCl concentration I have found to be more important for solubility depending on the size of the protein. Larger MW often needs more NaCl but normally I use it around 150-500mM. Often I will add up to 10% Glycerol for larger proteins to enhance stability. I also routinely add 0.5-1%Triton X100 to help with breakage and solubility, and Protease Inhibitors are generally used as welll. As for other additives care needs to be taken when using chelating and reducing agents. They can be used but you need to test them. Some of the issues can be gotten around (see other questions here for answers).
For a gentle lysis Lysozyme is good but the best complete lysis I believe is a cell disruptor, however some target proteins can aggregate badly when this is used. To enhance Dnase I have found addition of MgCl2 and CaCl2 to work. This also allows you to use EDTA in the lysis step with lysozyme incubation as once the cells are broken you can then add the metals with the Dnase which effectively binds to all the EDTA in the buffer allowing you to then add the lysis to your IMAC resin. IMAC resin also varies significantly so I would test a variety if you can. I have tried around 10-15 different companies and resins and you get a wide variety of results in yield, purity etc. All can be used but some require more optimisation. Imidazole in the lysis can reduce contaminant binding and 5-50mM can be used but test it at low concentration first or you may wash off your target.
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