Washing and Regenerating of Rho1D4 Agarose
This protocol delineates a regenerating procedure for PureCube Rho1D4 Agarose to allow for re-use of the matrix. Volumes are given in column bed volume (bv), i.e., 10 bv calls for 10 mL of buffer for a 1 mL column bed volume. Note that this protocol can be performed on a gravity flow column, but is much more convenient when done on an FPLC instrument. Reusability of PureCube Rho1D4 Agarose depends on many factors: the protein purified, buffer conditions, storage conditions, etc.. Therefore it is hard to predict how many times the matrix can be regenerated without significant decrease in performance.
Buffer compositions - Rho1D4 bead regeneration
High pH Regeneration Buffer, 500 mL
Component | Final concentration | Molecular weight (g/mol) | Stock concentration | Amount needed for stock | Stock needed for buffer |
---|---|---|---|---|---|
Tris-base, pH 8.5 | 50 mM | 121.14 | 1 M | 60.57 g/ 500 mL | 25 mL |
NaCl | 0.5 M | 58.44 | 5 M | 146.1 g/500 mL | 50 mL |
Instructions: Dissolve Tris base in 400 mL water, set the pH to 8.5 using HCl, and fill up to 500 mL. Use this stock solution to prepare the High pH Regeneration Buffer. |
Low pH Regeneration Buffer, 500 mL
Component | Final concentration | Molecular weight (g/mol) | Stock concentration | Amount needed for stock | Stock needed for buffer |
---|---|---|---|---|---|
Sodium acetate, pH 4.5 | 100 mM | 136.08 | 1 M | 68.04 g/ 500 mL | 50 mL |
NaCl | 0.5 M | 58.44 | 5 M | 146.1 g/500 mL | 50 mL |
Instructions: Dissolve sodium acetate in 400 mL water, set the pH to 4.5 using HCl, and fill up to 500 mL. Use this stock solution to prepare the Low pH Regeneration Buffer. |
A. | Procedure |
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Note: "bv" refers to column bed volume, i.e., 10 bv calls for 10 mL of buffer for a 1 mL column bed volume.
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