NHS-Activated Beads

NHS-Activated Beads are pre-activated agarose microspheres that can be directly used for coupling with amino-containing proteins or peptides. The pre-activated medium can be prepared into specialized affinity media as needed, enabling rapid and efficient one-step purification of target substances from complex systems. NHS-Activated Beads exhibit excellent pressure resistance and stable performance after protein coupling, making them suitable for large-scale industrial purification.

2.1 Preparation of Buffer Solutions

It is recommended to filter all water and buffer solutions through a 0.22 μm or 0.45 μm membrane before use.

Washing solution: 1 mM HCl

Coupling solution: 0.2 M NaHCO3, 0.5 M NaCl, pH 8.0

Blocking solution: 0.5 M ethanolamine, 0.5 M NaCl, pH 8.3 or 0.1 M Tris, pH 8.5

Washing solution 1: 0.1 M acetate-sodium acetate, 0.5 M NaCl, pH 3.0

Washing solution 2: 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.0

Preservation solution: 1X PBS containing 20% ethanol

Note: 1. The coupling solution can be selected from carbonate, phosphate, or other buffer systems without amino groups. Adding a certain concentration of salt ions to the buffer system can reduce non-specific adsorption.

2. When the coupling sample is an antibody, 1X PBS with 0.02% NaN3 or 1X PBS with 0.02% Proclin 300 can be used as the preservation solution.

2.2 Sample Preparation

Dissolve or dialyze the sample in the coupling solution, with a concentration of approximately 5-10 mg/ml.

2.3 Sample Coupling

The following example uses antibody coupling to purify antigens, introducing the coupling and subsequent purification steps.

1) Take an appropriate amount of NHS-Activated Beads, wash three times with the washing solution by suction filtration, and wash once with the coupling solution. Note: Do not dry the beads too much to avoid clumping. If clumping occurs, disperse by shaking or pipetting. Pre-cooled solutions can be used for rapid washing to reduce hydrolysis of pre-activated media.

2) Add the dissolved sample to the washed NHS-Activated Beads, with a volume ratio of NHS-Activated Beads to sample solution of approximately 1:1-2 (V/V).

3) Shake and react at 28°C for 2-4 hours or at 4°C overnight. Note: Ensure the beads are suspended; otherwise, coupling efficiency will be greatly affected.

4) After the reaction, collect the coupled sample for efficiency testing. Wash the beads with deionized water, add 2 times the column volume of blocking solution, and shake at 28°C for 1 hour.

Note: After coupling, the supernatant protein concentration cannot be measured by UV absorption. It is recommended to use electrophoresis or BCA quantification to test coupling efficiency.

5) Remove the reaction system, drain the blocking solution, wash the beads with 3 times the column volume of deionized water, and repeat washing with washing solution 1, deionized water, washing solution 2, and deionized water twice. Then store in an equal volume of preservation solution at 2-8°C.

2.4 Column Packing

2.4.1 Gravity Column Packing

1) Take a gravity column of appropriate specification, insert the lower frit, add an appropriate amount of pure water to rinse the column tube and frit, and close the lower outlet.

2) Mix the coupled beads evenly, pipette an appropriate amount of slurry into the gravity column, and open the lower outlet to drain the preservation solution.

3) Add an appropriate amount of pure water to rinse the medium. After the liquid in the column tube drains by gravity, close the lower outlet.

4) Insert the rinsed upper frit, ensuring no gaps between the frit and the beads and keeping it level.

5) The packed gravity column can directly add equilibration buffer for balancing.

2.4.2 Medium-Pressure Column Packing

Coupled beads can also be used for large-volume sample purification, involving various medium-pressure chromatography column packing methods. Below is a method for packing chromatography columns.

Before packing, calculate the column bottom area based on the column diameter, and calculate the required medium volume based on the desired packing height using the following formula:

V: Required medium volume (ml)

1.15: Compression coefficient

r: Column tube radius (cm)

h: Packing height (cm)

Note: The volume of the suspension taken should be twice the medium volume, as the medium volume only accounts for half of the total suspension volume, with the other half being the preservation solution.

1) Rinse the column bottom frit and connector with deionized water to ensure no bubbles on the frit. Close the column bottom outlet and leave 1-2 cm of deionized water at the bottom.

2) Suspend the beads and carefully pour the slurry continuously into the column. Pouring the slurry along the column wall with a glass rod can reduce bubble formation.

3) If using a reservoir, immediately fill the column and reservoir with water. Place the sample distributor on the slurry surface and connect it to the pump to avoid bubble formation in the distributor or inlet tube.

4) Open the column bottom outlet, start the pump, and run at the set flow rate. Initially, let the buffer flow slowly through the column, then gradually increase to the final flow rate to avoid hydraulic impact on the formed bed and ensure uniform packing. If the recommended pressure or flow rate cannot be achieved, use the maximum flow rate of your pump for good packing results. (Note: Do not exceed 75% of the maximum packing flow rate in subsequent chromatography procedures.) When the bed height stabilizes, run at least 3 times the bed volume of deionized water at the final packing flow rate. Mark the bed height.

5) Turn off the pump and close the column outlet.

6) If using a reservoir, remove it and place the distributor in the column.

7) Push the distributor to the marked bed height. Allow the packing solution to enter the distributor and tighten the distributor connector.

8) Connect the packed column to the pump or chromatography system and start equilibration. Adjust the distributor if necessary.

2.5 Sample Purification

2.5.1 Preparation of Buffer Solutions

It is recommended to filter all water and buffer solutions through a 0.22 μm or 0.45 μm membrane before use.

Equilibration/Wash buffer: 0.15 M NaCl, 20 mM Na2HPO4, pH 7.0

Elution buffer: 0.1 M glycine, pH 3.0

Neutralization buffer: 1 M Tris-HCl, pH 8.5

2.5.2 Incubation Purification

1) Based on the sample volume, add an appropriate amount of coupled beads to the column and drain the preservation solution by gravity.

2) Wash the beads with 5 times the medium volume of equilibration buffer and drain by gravity.

3) Add the sample, seal both ends of the column, and incubate at 4°C with shaking for 2-4 hours or at 37°C for 30 minutes to 2 hours.

4) After incubation, centrifuge or filter to collect the beads, retaining the supernatant as flow-through for electrophoresis analysis.

5) Wash the beads with 5 times the medium volume of wash buffer, centrifuge or filter to remove the supernatant, and repeat 3-5 times, changing tubes as needed.

6) Add 3-5 times the column volume of elution buffer for elution, incubate for 10-15 minutes, and collect the eluate by centrifugation or filtration. Repeat 2-3 times. Neutralize the eluate immediately, typically using 1/10 volume of neutralization buffer.

2.5.3 Gravity Column Purification

1) Equilibrate the packed gravity column with 5 times the column volume of equilibration buffer to condition the beads in the same buffer system as the target protein.

2) Load the sample onto the equilibrated gravity column, collect the flow-through, and repeat loading to increase binding efficiency.

3) Wash with 10 times the column volume of wash buffer to remove non-specifically adsorbed impurities, collecting the wash fractions.

4) Elute with 5 times the column volume of elution buffer, collecting fractions. Neutralize the eluate immediately, typically using 1/10 volume of neutralization buffer.

2.5.4 Medium-Pressure Column Purification

After packing, medium-pressure columns can be used with various conventional medium/low-pressure chromatography systems.

1) Fill the pump tubing with deionized water. Remove the upper cap, connect the column to the chromatography system, open the lower outlet, attach the pre-packed column, and tighten.

2) Rinse out the storage buffer with 3-5 times the column volume of deionized water.

3) Equilibrate the column with at least 5 times the bed volume of equilibration buffer.

4) Load the sample using the pump or sample loop.

Note: Increased sample viscosity can cause high backpressure even with small sample volumes. Do not exceed the column's binding capacity. Large sample volumes may also cause high backpressure, making injection difficult.

5) Wash the column with wash buffer until UV absorption reaches a stable baseline (typically at least 10-15 column volumes).

6) Elute with 5-10 times the column volume of elution buffer, collecting the eluate (target protein fractions). Neutralize the eluate immediately, typically using 1/10 volume of neutralization buffer.

After elution, rinse the beads with 5-10 column volumes of equilibration buffer, then 5-10 column volumes of pure water, and finally 2 column volumes of 20% ethanol. Store at 2-8°C.

2.6 SDS-PAGE Detection

Analyze the samples obtained from purification (including flow-through, wash, and elution fractions) and the original sample using SDS-PAGE to evaluate purification efficiency.