Preparation And Application Of Fe₃O₄ Magnetic Sub-microspheres Metal Chelating Affinity Medium

In this paper,solvent thermal synthesis of superparamagnetic Fe3O4sub-microspheres having superparamagnetic by epichlorohydrin,iminodiacetic acid modification,and transition metal ions chelated to prepare a superparamagnetic submicrospheres metal chelate affinity media,with 6xHis-Tag to the Bacillus subtilis 1-382 amino acid assay of recombinant proteins.The solvent thermal synthesis of superparamagnetic Fe3O4sub-microspheres have carried toward the superparamagnetic electron microscopy characterization can be seen syntheticsuperparamagnetic Fe3O4sub-microspheres microscopic uniform particle size stability in transmission and scanning electron.Using infrared MSF,MSF-IDA,MSF-IDA-Me were characterized prove IDA and Me has been modified to superparamagnetic Fe304sub-microspheres groups according to the location.The metal element analysis by ICP is further proof of the transition metal ion(Me)is connected to superparamagnetic Fe3O4sub-microspheres.Superparamagnetic Fe3O4sub-microspheres curve shows sub-microspheres towards its modification along little superparamagnetic influence,still maintain a good performance of supeiparamagnetic induction.By MSF,MSF-IDA,MSF-IDA-Cu2+,MSF-IDA-Ni2+ MSF-IDA-Co2+,MSF-IDA-Zn2+on the BSA,adsorption His-Tagprotein provedHMSF,MSF-IDA,MSF-IDA-Cu2+,MSF-IDA-Ni2+,MSF-IDA-Co2+,MSF-IDA-Zn2+ for non-His-Tag protein no adsorption;MSF,MSF-IDA for His-Tag protein and no adsorption on the His-Tag protein adsorption effect is MSF-IDA-Cu2+,MSF-IDA-Ni2+,MSF-IDA-Co2+,MSF-IDA-Zn2+four kinds of superparamagnetic sub-microspheres metal chelate affinity medium,and from protein electrophoresis may be preliminary to see miscellaneous almost no protein adsorption,the adsorption effect of the His-tag label protein is very good.For MSF-IDA-Me synthesis conditions and His-Tag protein adsorption optimized conditions.From experimental results,superparamagnetic Fe3O4 sub-microspheres step IDA modified after the activation,His-Tag highest concentration of protein adsorption IDA solution of 5%sodium bicarbonate buffer pH 9.5.When MSF-IDA chelating transition metal ions MSF-IDA-Me,a transition metal ion concentration 0.1 mol/L to achieve a saturated solution.When MSF-IDA-Me of His-Tag protein adsorption,the reaction no longer than 2min,the reaction has been carried out thoroughly.Analysis of His-Tag protein was eluted adsorption capacity.Seen MSF-IDA-Cu2+,MSF-IDA-Ni2+,MSF-IDA-Co2+,MSF-IDA-Zn2+adsorptio n capacity of the His-Tag protein was MSF-IDA-Cu2+>MSF-IDA-Ni2+>MSF-IDA-Co2+>MSF-IDA-Zn2+.MSF-IDA-Cu2+,MSF-IDA-Ni2+ adsorption His-Tag protein is difficult to be eluted from the media.MSF-IDA-Co2+,MSF-IDA-Zn2+ adsorption His-Tag protein more likely to be separated.Study MSF-IDA reload times of transition on His-Tag protein purification effect after metal ions.Seen MSF-IDA-Co2+,MSF-IDA-Zn2+loaded on a transition metal ion,His-Tag protein adsorption,elution after the operation 6-7 times,media separation ability of His-Tag protein was reduced to about 80%.After MSF-IDA heavy transition metal ions on the role of His-Tag protein was purified with the original media is quite basic,medium reloading three times purified His-Tag protein capacity decreased to about 85%.