Description: Nickel Resin, Nickel agarose, Fast Flow
Reagent: Nickel charged cross-linked 6% agarose. The Nickel resin is supplied as 50% slurry in 20% Ethanol.
Recommended usage: Nickel Resin can be used for purifying recombinant protein with His tag at either N-terminus or C-terminus (minimum 6xHis). In theory, Nickel resin can also be used to purify protein with an internal His tag but this has not been validated by the manufacturer. The binding capacity for the resin tested is about 10-20mg of His tagged protein per mL of resin. If the starting material is culture sup, you can easily achieve 95% purity with one-step purification using Nickel resin from BioTimes.
Vial Size: 10mL, 50mL
Chemical stability: 1M NaOH, 6M guanidine hydrochloride, 20% ethanol, 8M urea, commonly used aqueous buffers for Nickel purification
Storage and Handling: Store the vial at 4°C (DO NOT FREEZE). The unopened vial is stable for twelve months when stored at 4°C.
Application: Protein purification, Immunoprecipitation
Recommended protocol: Nickel resin can be used for His-tagged recombinant protein purification in the batch or column method.
Resin Preparation: thoroughly resuspend the Nickel resin by gentle inversion until the resin is in uniform suspension. Apply the desired volume of the resin into the empty column and allow the beads to be settled down and liquid drained. Do not let the resin run dry. Apply at least 2 column volumes (net resin volume) of Ni-A buffer (20mM Tris, 300mM NaCl, pH8) to wash off the ethanol. Now the resin is ready for use.
Binding and washing: for column purification, we recommend running cell lysate or culture sup at the speed of 0.5-1.0 mL/min. For batch purification, the washed Ni resin can be added to the cell lysate or culture sup and stirred with low speed (50-70 rpm) on the stir plate or rotating on the rotator. The binding usually can be completed at room temperature for 3-5 hours or overnight at 4°C. A low concentration of Imidazole (10-40 mM) can be added to the cell lysate or culture sup before loading the sample with Ni resin. After the batch binding, the sample with the resin can be loaded into Bio-rad Econo-column. After the resin is completely settled down (for batch purification) or after the lysate or sup is completely loaded into the column (for column purification), wash the column with Ni-A buffer to get rid of non-specific binding. This can be monitored by the Bradford method or UV monitor till it reaches the baseline. In some cases, it is beneficial to wash the resin with a low concentration of Imidazole (20mM, 40mM up to 100mM Imidazole in Ni-A buffer) to get rid of non-specific binding (titration experiment is needed for optimizing the purification of each target protein).
Target Protein Elution: Bound protein can be eluted with 200-500 mM Imidazole in Ni-A buffer (elution concentration should be optimized for each target).
Resin cleaning: After each use, wash the column with 5 column volume of the deionized water. Cleaning with 1M NaOH at 1-2 mL/min for about 20 minutes. Equilibrate the resin with 5-10 column volume of Ni-A buffer. 
Resin regeneration:  The resin can be regenerated with the following steps:
6M guanidine-HCl (2-5 column volume) at 1-2mL/min (washing)
100mM EDTA (2-5 column volume) at 2-5mL/min (stripping)
Deionized water (5-10 column volume) at 5-10 mL/min
0.1M NiCl2 (2-5 column volume) at 1-2mL/min (charging)
Deionized water (5-10 column volume) at 5-10 mL/min
Ni-A buffer (5-10 column volume) at 5-10 mL/min
20% Ethanol (5-10 column volume) at 5-10 mL/min (optional for long term storage)
Resin storage: after the cleaning or regeneration (can be reused at least 10 times without obvious loss in binding capacity), transfer the resin into the appropriate container and store it at 4°C. Nickel resin is stable in Ni-A buffer for 1-2 months at 4°C. For long-term storage, we recommend storing the resin in 20% Ethanol at 4°C.