rProtein L Beads 4FF
rProtein L Beads 4FF is an affinity chromatography medium designed for purification of classes, subclasses and fragments of immunoglobulins from biological fluids and from cell culture media. The recombinant protein L ligand is immobilized to highly cross-linked 4% agarose beads. The Characteristics of rProtein L Beads 4FF are summarized in Table 1.
Protein L is recombinantly expressed in E. coli and retains the property of binding to the antibody κ-chain without affecting the antigenic binding site of the antibody. Protein L binds well to the kappa light chain of human and mouse, and may bind specifically to certain kappa isoforms of other species. Protein L binds weakly to rabbit immunoglobulins and does not bind bovine, goat or sheep source immunoglobulins.
Table 1. Characteristics of rProtein L Beads 4FF
Item | Description |
Matrix Spherical | Highly cross-linked 4% agarose beads |
Ligand | recombinant protein L |
Static Binding Capacity | ＞15mg Mouse lgG/ml medium |
Particle size | 45-165 μm |
Maximum Pressure | 0.3 MPa, 3 bar |
pH | 3-10 |
Storage Solution | 1×PBS containing 20% ethanol |
Storage Temperature | 2-8℃ |
2.1 Buffer Preparation
Water and chemicals used for buffer preparation should be of high purity. It is recommended filtering the buffers by passing them through a 0.22 or 0.45 μm filter before use.
Binding/Wash Buffer: 0.15 M NaCl, 20 mM Na2HPO4, pH 7.0
Elution Buffer: 0.1 M glycine, pH 3.0
Neutralization Buffer: 1 M Tris-HCl, pH 8.5
2.2 Sample Preparation
To ensure 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.
It is recommended filtering the sample solution by passing them through a 0.22 μm or 0.45 μm filter before use.
2.3 Packing Columns
rProtein L Beads 4FF is easy to pack and use, and its high flow properties make it excellent for industrial scaling-up. Here we describe the packing procedure of rProtein L Beads 4FF to medium pressure chromatography columns.
1) Remove air from the column dead spaces by flushing the end-piece and adapter with packing buffer. Make sure no air has been trapped under the column net.
2) Close the column outlet leaving the net covered with packing buffer.
3) Resuspend the beads stored in its container by shaking (avoid stirring the sedimented medium). Pouring the slurry down a glass rod
held against the column wall will minimize the introduction of air bubbles.
If using a packing reservoir, immediately fill the remainder of the column and reservoir with packing buffer. Mount the adapter or lid of the
packing reservoir and connect the column to a pump. Avoid trapping air bubbles under the adapter or in the inlet tubing.
4) Open the bottom outlet of the column and set the pump to run at the desired flow velocity. Ideally, rProtein L Beads 4FF is packed at a constant pressure of approximately 3 bar (0.3 MPa). If the recommended pressure or flow velocity can not be obtained, use the maximum flow velocity the pump can deliver. This should also give a reasonable well-packed bed. Do not exceed 75% of the packing flow velocity in subsequent chromatographic procedures.
5) When the bed has stabilized, close the bottom outlet and stop the pump.
If using a packing reservoir, disconnect the reservoir and fit the adapter to the column. If using the column, carefully place the top filter on top of the bed before fitting the adapter.
6) With the adapter inlet disconnected, push the adapter down, approximately 2 mm into the bed, allowing the packing solution to flush the adapter inlet.
7) Connect the pump, open the bottom outlet and continue packing. The bed will be further compressed at this point and a space will be formed between the bed surface and the adapter.
8) Close the bottom outlet. Disconnect the column inlet and lower the adapter approximately 2 mm into the bed. Connect the pump. The column is now ready to use.
2.4 Sample Purification
1) Fill the syringe or pump tubing with binding buffer. Remove the stopper and connect the column to the syringe (with the provided connector), or pump tubing, “drop to drop” to avoid introducing air into the column. Remove the snap-off end at the column outlet.
2) Wash the column with 10 column volumes of binding buffer .
3) Apply the sample, using a syringe fitted to the connector or by pumping it onto the column.
4) Wash with 5 to 10 column volumes of binding buffer or until no material appears in the effluent.
5) Elute the sample with 5-10 column volumes Elution Buffer. Collect the eluate containing the target immunoglobulin and immediately neutralize to pH 7.4 with Neutralization Buffer (1/10 volume of total eluate).
6) Equilibrate the column with 3 column volumes of Binding Buffer, 5 column volumes of distilled water and 5 column volumes of 1×PBS containing 20% ethanol. Finally store the resin with 1×PBS containing 20% ethanol at 4℃.
2.5 Analysis
Identify the fractions using UV absorbance, SDS-PAGE, or western blot.
In general, rProtein L Beads 4FF are well suited for reuse a number of times. When precipitation and protein aggregation cause the loss of velocity and combined loads, you need to clean the medium.
Remove the precipitation or denatured protein
Wash the column with 2 column volumes 6 M guanidine hydrochloride solution. Finally wash the column with 5 column volumes 1×PBS (pH 7.4).
Remove the hydrophobically bound protein
Wash the column with 3-4 column volumes 70% ethanol or 2 column volumes 0.1% non-ionic detergent. Finally wash the column with 5 column volumes 1×PBS (pH 7.4).
Problem | Possible Cause | Solution |
The flow rate of the column is very low. | The sieve plate is blocked. | Clean or replace the sieve plate. |
Column is clogged. | Cleaning in place(part 3). | |
Filtering the sample solution by passing them through a 0.22 μm or 0.45 μm filter. | ||
The curve is not stable during sample purification | Tiny air bubbles from buffer or sample. | De-gas buffers and samples. Do not allow the column to dry. |
A considerable amount of sample has been loaded, but no specific antibody of interest is detected. | The concentration of antibody of interest is very low. | Purify the antibody using the specific antigen coupled to a beads (i.e., PabPur Sulfolink Beads, Cat. No. SA018005). |
The antibody is sensitive to low-pH elution buffer | Neutralize the eluted fractions with Neutralization Buffer immediately after elution. | |
The recovery rate gradually decreases. | The sample is overloaded. | Reduce the loading volume. |
The column is too dirty and the binding capicity is reduced. | Cleaning in place (part 3). |
Product | Cat. No. | Size |
rProtein L Beads 4FF | SA033005 SA033025 SA033100 SA033500 SA03301L SA03310L | 5 ml 25 ml 100 ml 500 ml 1 L 10 L |
