Important Product Information
• Protein yield and purity are dependent upon the expression level, conformation and solubility characteristics of the recombinant fusion protein. Therefore, it is important to optimize these parameters before attempting a large-scale purification. For best results, perform a small-scale test to estimate the expression level and determine the solubility of each His-tagged protein.
• Optimization of the lysis procedure is critical for maximizing protein yield. Some methods for protein extraction include using B-PER Bacterial Protein Extraction Reagent, mechanical methods such as freeze/thaw cycles, sonication or French press.
• Sometimes overexpressed proteins are sequestered in inclusion bodies. Inclusion bodies of His-tagged proteins can be solubilized in 8M urea, 6M guanidine) and purified with the Ni-NTA resin, but a denaturant must be added to buffers so the protein remains soluble throughout the procedure.
• These instructions are effective for many types of samples; however, optimization might be required to further reduce nonspecific binding. To optimize conditions, adjust the recommended imidazole concentration in the Equilibration, Wash and Elution Buffer.
• Avoid using protease inhibitors or other additives that contain chelators, such as EDTA, or strong reducing agents, such as DTT or β-mercaptoethanol, which will disrupt the function of the nickel resin.
• When using the Bradford Assay to monitor protein concentration in the elution fractions, dilute the samples at least 1:2 before performing the protein assay.