2'5' ADP Sepharose 4B

• Cat#No#: 2'-387C
• Product Overview: 2'5' ADP Sepharose 4B resin with immobilized NADP structural analog is primarily used for purification of enzymes requiring NADP as a cofactor. 2'5' ADP is an NADP structural analog immobilized on Sepharose 4B. It binds and immobilizes enzymes requiring this cofactor.
• Description: 2'5' ADP Sepharose 4B interacts strongly with NADP+-dependent dehydrogenases. Selective elution with gradients of NAD+ or NADP+ has allowed the resolution of complex mixtures of dehydrogenase isoenzymes using 2'5' ADP Sepharose 4B.
• Available capacity: approx. 0.4 mg glucose-6-phosphate dehydrogenase/ml drained medium.
• Medium Preparation: Weigh out the required amount of freeze dried powder (1 g freezedried powder gives 3.5 to 5 ml final medium volume) and suspend it in distilled water. The medium swells immediately and should now be washed for 15 minutes with distilled water on a sintered glass filter. Use approximately 200 ml distilled water per gram freeze-dried powder, added in several aliquots.
  Prepare a slurry with binding buffer, in a ratio of 75% settled medium to 25% buffer. The binding buffer should not contain agents which significantly increase the viscosity. The column may be equilibrated with viscous buffers at reduced flow rates after packing is completed.
• Ligand Coupling Method: Cyanogen bromide activation
• Packing Column: 1. Equilibrate all material to the temperature at which the chromatography will be performed.
  2. De-gas the medium slurry.
  3. Eliminate air from the column dead spaces by flushing the end pieces with buffer. Make sure no air has been trapped under the column net. Close the column outlet with a few centimeters of buffer remaining in the column.
  4. Pour the slurry into the column in one continuous motion. Pouring the slurry down a glass rod held against the wall of the column will minimize the introduction of air bubbles.
  5. Immediately fill the remainder of the column with buffer, mount the column top piece onto the column and connect the column to a pump.
  6. Open the bottom outlet of the column and set the pump to run at the desired flow rate.
  7. Maintain the packing flow rate for 3 bed volumes after a constant bed height is reached.
• Matrix: Agarose, 4%
• Water Regain: Swollen medium should be stored in neutral pH at 2ºC to 8ºC in presence of a bacteriostat, e.g., 20% ethanol. Exposure to solutions with pH greater than 10 may cause loss of phosphate groups. The swollen medium must not be frozen.
• Particle Size: 45 µm-165 µm
• Average particle size: ~90 µm
• Ligand density: approx. 2 µmol 2'5' ADP/ml drained medium.
• Recommended flow rate: < 75 cm/h at 25 °C, HR 16/10 column, 5 cm bed height.
• Chemical stability: Stable to commonly used aqueous buffers and additives like detergents.
• Physical stability: Negligible volume variation due to changes in pH or ionic strength.
• pH working range: 4–10
• CIP stability: 4–10
• Temperature stability: 2 to 8°C
• Cleaning-in-place: In some applications, substances like denatured proteins or lipids do not elute in the regeneration procedure. These can be removed by washing the column with a detergent solution at 37ºC for one minute.
• Pack size: 5 g
• Maximum flow velocity: 75 cm/h at 25ºC