Protein L Resin

High temperature heating is not recommended. The agarose melts above 65°C.

Water and chemicals used for buffer preparation should be of high purity. It is recommended filtering the buffers by passing them through a 0.45 μm filter before use.
Binding/Wash Buffer: 20 mM Na2HPO4, 0.15 M NaCl, pH 8.0
Elution Buffer: 0.1 M glycine, pH 2.5
Neutralization Buffer: 1 M Tris-HCl, pH 8.5

To insure that proper ionic strength and pH are maintained for optimal binding, it is necessary to dilute serum samples, ascite fluid or cell culture supernatant at least 1:1 with Binding/Wash Buffer. Alternatively, the sample may be dialyzed overnight against Binding/Wash Buffer.

Packing of Column
1) Resuspend completely the resin and transfer 1 ml slurry to a new column, in which 1 ml Binding/Wash Buffer was added in advance.
2) Allow the resin to settle down and the buffer to drain from the column.
3) Add 5 ml binding/Wash Buffer onto the column to equilibrate the resin and drain the buffer with a flow speed of about 1 ml/min.

Column Purification
1) Apply the sample onto the column and drain the flow-through with a flow speed of about 1 ml/min. Collect the flow-through for measuring the binding efficiency to the resin, i.e. by SDS-PAGE.
2) Wash the column with 30 ml Binding/Wash Buffer and drain the buffer with a flow speed of about 2 ml/min, or until the absorbance of the effluent at 280 nm is stable.
3) Elute the immunoglobulins with 10-15 ml Elution Buffer and drain the eluate with a flow speed of about 1 ml/min. Collect the eluate and immediately neutralize to pH 7.4 with Neutralization Buffer (1/10 volume of total eluate)

Regeneration of Column
Regenerate the column by washing the resin with 10 ml Elution Buffer followed by equilibration with 5 ml Binding/Wash Buffer. Columns can be regenerated up to 10 times without significant loss of binding capacity.