Streptavidin Agarose 6FF

1. Recommended Buffers (Not Provided)

Note: It is recommended to prepare the buffers using high-purity water and sterilize them by filtration through a 0.45 μm membrane after preparation.

2. Sample Preparation

1) Before loading the sample onto the column, centrifuge the sample or filter it through a 0.22 μm or 0.45 μm membrane to reduce impurities and prevent column clogging.

2) Before loading, dilute or dialyze samples such as serum, ascites, or cell culture medium against the equilibration/wash buffer to ensure that the sample solution has an appropriate ionic strength and pH.

3. Packing of Streptavidin Agarose 6FF

Before use, thoroughly resuspend and mix the Streptavidin Agarose 6FF before pipetting.

3.1 Packing a Gravity-Flow Column

Rinse the upper and lower column frits before using the gravity-flow column.

1) Select a gravity-flow chromatography column of an appropriate size and install the lower frit. Rinse the column and frit with ddH₂O, and then close the bottom outlet.

2) Transfer an appropriate volume of the Streptavidin Agarose 6FF suspension into the gravity-flow column. Open the bottom outlet and allow the storage solution to drain completely.

Notes:

a) Monitor the flow rate of the storage solution and add additional Streptavidin Agarose 6FF to the column in time, if necessary.

b) Avoid the formation of gaps within the packed resin.

3) Add an appropriate volume of ddH₂O to wash the resin. After the liquid in the column has drained completely, close the bottom outlet.

4) Install the pre-rinsed upper frit into the column.

Note: Avoid leaving gaps between the frit and the packed resin.

5) The packed gravity-flow column can be equilibrated directly with equilibration buffer. If the column will not be used immediately, add storage solution containing 20% ethanol in 1× PBS and store it at 2-8°C.

3.2 Packing a Medium-Pressure Chromatography Column

This product is also suitable for use with various medium-pressure chromatography columns. Before packing, calculate the cross-sectional area of the column according to the column radius, and calculate the required resin volume according to the desired packed-bed height using the following equation:

V = 1.15πr²h

V: Required resin volume, mL

1.15: Compression factor

r: Column radius, cm

h: Desired packed-bed height, cm

Note: The settled resin accounts for half of the total suspension volume. Therefore, the required suspension volume should be twice the calculated resin volume.

Pack the medium-pressure chromatography column according to the following procedure:

1) Rinse the column frit and connectors with ddH₂O. Ensure that no air bubbles remain on the bottom frit, close the bottom outlet, and leave 1-2 cm of ddH₂O at the bottom of the column.

2) Thoroughly mix the Streptavidin Agarose 6FF and carefully pour the suspension continuously into the chromatography column.

Note: Pouring the suspension along the column wall with the aid of a glass rod can reduce bubble formation.

3) If a reservoir is used, immediately fill both the chromatography column and reservoir with water. Position the flow adaptor on the surface of the suspension and connect it to the pump.

Note: Avoid introducing air bubbles into the flow adaptor or sample tubing.

4) Connect the chromatography column to the pump, open the bottom outlet, and start the pump at a flow rate of approximately 0.5-1 mL/min. The flow rate may be adjusted as appropriate. Initially allow the buffer to flow slowly through the column, and then gradually increase it to the final flow rate. This helps prevent hydraulic pressure from disturbing the packed bed and reduces uneven bed formation. Once the packed-bed height has stabilized, pass at least 3 packed-bed volumes of ddH₂O through the column at the final packing flow rate. Mark the packed-bed height.

Notes:

a) If the recommended pressure or flow rate cannot be achieved, the maximum flow rate of the pump may be used to obtain satisfactory packing performance.

b) During chromatography, the buffer flow rate should not exceed 75% of the maximum packing flow rate.

5) Stop the pump and close the column outlet.

6) If a reservoir is used, remove the reservoir and place the flow adaptor into the chromatography column.

7) Push the flow adaptor down to the marked packed-bed height. Allow liquid from the column to enter the adaptor, and then tighten the adaptor connector.

8) Connect the packed chromatography column to the pump or chromatography system and equilibrate the column.

Note: The position of the flow adaptor may be readjusted as needed.

4. Sample Purification

Sample purification can be performed using the centrifugation method, gravity-flow column method, or medium-pressure chromatography column method.

4.1 Centrifugation Method

1) According to the amount of sample to be purified, transfer an appropriate volume of the Streptavidin Agarose 6FF suspension into a centrifuge tube. Centrifuge at 1,000 rpm for 1 min and discard the supernatant.

2) Add 5 settled-resin volumes of equilibration buffer, centrifuge at 1,000 rpm for 1 min, and discard the supernatant. Repeat the washing step twice.

3) Add the sample, close the centrifuge tube, and incubate at 4°C for 2-4 h or at 37°C for 0.5-2 h.

4) After incubation, centrifuge at 1,000 rpm for 1 min and discard the supernatant. The supernatant may be retained as the flow-through fraction for electrophoretic analysis.

5) Wash the resin with 5 settled-resin volumes of wash buffer. Centrifuge at 1,000 rpm for 1 min and discard the supernatant. Repeat the washing step 3-5 times.

Note: Avoid aspirating the resin when removing the supernatant. It is recommended to transfer the resin to a new EP tube during the washing procedure.

6) Elute with 3-5 settled-resin volumes of elution buffer. Incubate at room temperature for 5 min and centrifuge at 1,000 rpm for 1 min. The elution step may be repeated 2-3 times.

7) Wash the resin with 5-10 settled-resin volumes of equilibration buffer, followed by 5-10 settled-resin volumes of ddH₂O. Finally, wash with 2 settled-resin volumes of 20% ethanol and store at 2-8°C.

4.2 Gravity-Flow Column Method

1) Column Equilibration: Equilibrate the packed Streptavidin Agarose 6FF gravity-flow column with 5 column volumes of equilibration buffer. Repeat the equilibration step 2-3 times.

2) Sample Loading: After adding the sample, allow it to remain in the column for at least 2 min to ensure sufficient contact between the sample and the resin. Collect the flow-through.

Note: Repeated sample loading can improve binding efficiency.

3) Washing: Wash the column with 10-15 column volumes of wash buffer to remove nonspecifically adsorbed contaminating proteins. Collect the wash fraction.

4) Elution: Elute with 5-10 column volumes of elution buffer. Collect the eluate in separate fractions, with each tube containing one column volume, and analyze each fraction separately. This helps ensure complete elution of the target protein while maintaining an appropriate protein concentration and purity.

5) Wash the resin with 5-10 column volumes of equilibration buffer, followed by 5-10 column volumes of ddH₂O. Finally, wash with 2 column volumes of 20% ethanol and store at 2-8°C.

4.3 Medium-Pressure Chromatography Column Method

1) Fill the pump tubing with ddH₂O. Remove the upper stopper, connect the chromatography column to the chromatography system, open the bottom outlet, connect the prepacked column to the system, and tighten the connection.

2) Wash the column with 3-5 column volumes of ddH₂O.

3) Equilibrate the chromatography column with ≥5 column volumes of equilibration buffer.

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

Notes:

a) Do not exceed the binding capacity of the column.

b) Increased sample viscosity or volume may cause increased back pressure in the chromatography column.

5) Wash the column with ≥10-15 column volumes of wash buffer until the UV absorbance reaches a stable baseline.

6) Elute with 5-10 column volumes of elution buffer. The collected eluate contains the target protein.

7) Wash the resin with 5-10 column volumes of equilibration buffer, followed by 5-10 column volumes of ddH₂O. Finally, wash with 2 column volumes of 20% ethanol and store at 2-8°C.

5. SDS-PAGE Analysis

Evaluate the purification efficiency by SDS-PAGE using the original sample, flow-through fraction, wash fraction, and elution fraction.

Note: Guanidine hydrochloride is highly charged and can neutralize the charge contributed by SDS. It may therefore affect the charge of proteins in the loading buffer and cause precipitation during electrophoresis. The samples may be treated by dialysis or salting-out. For dialysis, place the sample in a dialysis bag and use PBS as the dialysis buffer. Dialyze twice for 1 h each time. A dialysis-buffer volume 100 times the sample volume is recommended. To reduce protein precipitation caused by rapid changes in pH, 1 M Tris, pH 9.0, may be added to neutralize the sample.

6. Resin Cleaning

This product can be reused without regeneration. However, as nonspecifically bound proteins and protein aggregates accumulate, the binding capacity and flow rate of the resin may decrease significantly. The resin should therefore be cleaned according to the following methods: