Protein L Resin Bead
- Peptostreptococcus magnus
- Affinity Chromatography (AC), Purification (Purif)
- Protein L Resin is an affinity chromatography medium designed for easy, one-step purification of classes, subclasses and fragments of immunoglobulins from biological fluids and from cell culture media.
- Ligand Highly purified protein L
Number of Ig binding sites per ligand 5
MW of ligand Approximately 42 kDa
PI of ligand 4.57
Degree of substitution Approximately 2 mg protein L/ml settled resin
Static binding capacity >15 mg rabbit IgG/ml settled resin
Matrix spherical Agarose, 4% cross-linked
Average particle size 90 μm (45 - 165 μm)
Storage solution 1×PBS containing 20% ethanol
Sanitization Washing of the packed column with 70% ethanol
- The highly purified protein L ligand is coupled to 4% highly cross-linked agarose. The coupling is optimized to give high binding capacity for immunoglobulins. The static binding capacity of Protein L Resin is greater than 15 mg rabbit IgG/ml settled resin. The dynamic binding capacity will vary depending on several factors such as target antibody, flow rate etc.
- Bead Ligand
- Protein L
- Bead Matrix
- Agarose beads
- Bead Size
- 90 µm
High temperature heating is not recommended. The agarose melts above 65°C.
- Reagent Preparation
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
- Sample Preparation
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.
- Assay Procedure
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.
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.
- For Research Use only
- 4 °C
- Storage Comment
- Store regenerated Protein L Resin in Binding/Wash Buffer containing 20% ethanol at 2°C to 8°C. Do not freeze.
- Expiry Date
- 18 months
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- Protein L, first isolated from the surface of bacterial species Peptostreptoccus magnus, binds immunoglobulin through κ light chain interaction. Protein L binds a wider range of Ig classes and subclasses than other antibody-binding proteins such as protein A or protein G. It can bind to all classes of Ig (i.e., IgG, IgM, IgA, IgE, and IgD) and also the single chain variable fragments (Scfv) and Fab fragments.