Equipment:
Affinity chromatography column (Bio-Rad 731-1550 Poly-Prep column, 0.8×4 cm)
Collector system (approximately 25 additional clean tubes are needed for fraction collection)
Reagents:
Metal Chelate Affinity Chromatography Beads (Ni-NTA agarose, QIAGEN 30210) – 1 mL
#The nitrilotriacetic acid (NTA) groups are covalently linked to the cross-linked agarose matrix and bind nickel ions. The beads are pre-saturated with Ni²+ ions before use.
Buffer B-300/0: 50 mM Na₃PO₄, pH 8.0; 0.3 M NaCl
#Add 10 mM β-mercaptoethanol just before use to maintain protein integrity.
Buffer B-300/20: Buffer B-300/0 with 20 mM imidazole added
Buffer B-300/250: Buffer B-300/0 with 250 mM imidazole added
Procedure:
1. The Ni-NTA agarose (1 mL) has already been packed into the column, forming a light blue gel. Place the column in a vertical column holder on an iron stand. Remove the packing material from the bottom, then rinse the column with 20 mL of Buffer B-300/0. Close the outlet to prepare for sample loading.
2. Carefully remove the buffer above the gel surface. Take the sample (IEX) out of the refrigerator and bring it to room temperature. Slowly pipette the sample onto the top of the gel, avoiding disturbance to the gel bed. Allow the sample to flow through the column while collecting fractions in 2.5 mL aliquots per tube.
3. Once all the sample has entered the column, close the outlet valve. Then, slowly add Buffer B-300/20 and open the outlet to collect the eluate. Continue washing the column with 30 mL of Buffer B-300/20.
4. Next, wash the column again with 30 mL of Buffer B-300/250 using the same procedure, and collect the eluate.
5. After all fractions have been collected, measure the protein concentration and enzyme activity for each fraction. Select the fractions with the highest enzyme activity, combine them, and record the total volume (AF). Reserve 100 μL of the purified sample for further analysis or storage.
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