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How to purify my GST-fusion Recombinant Protein with the GSTrap-Kit
GST fusion proteins can be purified directly from bacterial lysates with a one-step method using GSTrap. The fusion proteins are eluted under mild, non-denaturing conditions that preserve protein antigenicity and function. Download the complete GSTrap methodbook to read more.
Operation
The columns can be operated with a syringe, peristaltic pump or a liquid chromatography system.
Buffer preparation
Water and chemicals used for buffer preparation should be of high purity. We recommend filtering the buffers by passing them through a 0.45 µm filter before use.
Binding buffer: PBS pH 7.3 (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 , 1.8 mM KH2 PO4 , pH 7.3)
Elution Buffer: 50 mM Tris ™ -HCl, 10 mM reduced glutathione, pH 8.0
Sample preparation
The sample should be centrifuged and/or filtered through a 45 µm filter before it is applied to the column. If the sample is too viscous, dilute it with binding buffer to prevent clogging the column.
Purification
1. Fill the pump tubing or syringe with binding buffer. Connect the column to the syringe (use the adaptor supplied) or pump tubing “drop to drop“ to avoid introducing air into the column.
2. Remove the twist-off end.
3. Equilibrate the column with 5 column volumes of binding buffer.
4. Apply the sample using a syringe fitted to the luer adaptor or by pumping it onto the column. For best results, use a flow rate of 0.2–1 ml/min (1 ml column) and 1–5 ml/min (5 ml column) during sample application.
5. Wash with 5–10 column volumes of binding buffer or until no material appears in the effluent. A flow rate of 1–2 ml/min (1 ml column) and 5–10 ml/min (5 ml column) is recommended for washing.
6. Elute with 5–10 column volumes of elution buffer. A flow rate of 1–2 ml/min (1 ml column) and 5–10 ml/min (5 ml column) is recommended for elution.
Note:
One of the most important parameters affecting the binding of GST fusion proteins or other glutathione binding proteins to GSTrap is the flow rate. Due to the relatively slow binding kinetics between GST and glutathione, it is important to keep the flow rate low during sample application for maximum binding capacity.
Volumes and times used for elution may vary among fusion proteins. Additional elutions with higher concentrations of glutathione may be required. Flow-through, wash and eluted material from the column should be monitored for GST fusion proteins using SDS-PAGE in combination with Western Blot if necessary.
The GST Detection Module can be used to optimize conditions for elution or to trace steps in the purification of a GST fusion protein. The Module is designed to identify GST fusion proteins using either a biochemical or an immunological assay (3,4).
The concentration of GST fusion protein can be estimated by measuring the absorbance at 280 nm. The GST tag can be approximated using the conversion; A 280 _ 1 corresponds to ~ 0.5 mg/ml.
The concentration of GST fusion protein may also be determined by standard chromogenic methods (e.g. Lowry, BCA, and Bradford assays). If Lowry or BCA assays are to be used, the sample must first be buffer exchanged using a HiTrap Desalting column or dialysed against PBS to remove glutathione, which can interfere with the protein measurement. The Bradford method can be used in the presence of glutathione.
The reuse of GSTrap depends on the nature of the sample and should only be performed with identical samples to prevent cross-contamination. |
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