Sepharose CL-2B, 10 L
Sepharose CL-2B is a well-proven cross-linked agarose gel filtration base matrix and is frequently used for coupling affinity ligands to the matrix. The matrix is not pre-activated and the user performs all steps in coupling.
FAQ
Change Control Notifications and Regulatory Support Documentation are available for this product at the Regulatory Support Web Application, www.cytivalifesciences.com/rsf
After registering an account, you can start your subscription free of charge.
The column packing method described below has two steps. Resolution increases with increased bed height in size exclusion. If higher resolution is important, it is preferable to choose a bed height of at least 60 cm.
Instruments needed for packing:
Pump
Recommended columns: XK column series from Cytiva with one or two adapters
Packing reservoir (empty XK glass tube of same dimension as column)
Packing connector
- Prepare the chromatography medium by making a slurry with the eluent buffer in a ratio of 75% settled gel to 25% buffer. Degas the slurry before packing.
- Mount a packing reservoir, using the packing connector, at the top of a column.
- Insert an end piece or and adapter at the bottom of the column.
- Mount the column with the packing reservoir vertically on a laboratory stand. Eliminate air from the column dead spaces by flushing the end piece or adapter with eluent or distilled water. Make sure no air has been trapped under the net. Close the column outlet with approximately one centimeter of eluent remaining in the column.
- Pour the slurry in a single operation. Pouring the slurry down a glass rod held against the wall of the column will minimize the introduction of air bubbles. Fill the reservoir to the top with eluent or distilled water.
- Screw on the reservoir cap tightly and connect it to the pump.
- Open the bottom outlet of the column and start the pump at the flow given for step 1 in the table below.
- Pack the gel until the gel bed has reached a constant height.
- Stop the pump, close the column outlet and remove the packing connector and reservoir.
- Remount the column on the stand. Carefully fill the rest of the column with eluent to form an upward meniscus.
- Insert the adapter at an angle into the column, ensuring that no air is trapped under the net.
- Slide the plunger slowly down the column so that the air above the net and in the capillary tubings is displaced by eluent.
- Lock the adapter in position on the gel surface. Open the column outlet and apply a constant pressure given for step 2 in the table, until the gel bed has reached a constant height.
- Mark on the column the position of the bed surface, stop the pump, close the column outlet and adjust the adapter to the bed surface and then push the adapter a further 3-4 mm.
The column is now ready to use.
Recommended packing flow rates and pressures:
Chromatography medium | Step 1 Linear flow rate (cm/h*) |
Step 2 Pressure (MPa, bar, psi) |
---|---|---|
Sepharose CL-2B | 30 | 0.02, 0.2, 2.8 |
Sepharose CL-4B | 30 | 0.025, 0.25, 3.6 |
Sepharose CL-6B | 30 | 0.045, 0.45, 6.4 |
*Volumetric flow rate (ml/min)= [Linear flow rate (cm/h) × column cross-sectional area (cm2)]/60.