Thiopropyl Resin for the purification of thiol group containing proteins
Thiopropyl resin is a helpful tool for purifying thiol-containing proteins by covalent chromatography. When thiol-containing molecules come in contact with the thiopropyl resin, the target molecules covalently react with the resin support, and a covalent bond is created between the target molecule and the resin support, releasing 2-thiopyridone. This absorbs at ~ 343nm, and the release of 2-thiopyridone can indicate successful immobilization of thiol-containing proteins and molecules.
The newly formed covalent bond is reversible and can be broken with reducing agents.
Covalent bond chromatography is useful for separating thiol-containing molecules, proteins, or enzymes from non-thiol-containing proteins. Purification of enzymes with active-site thiol groups from denatured enzymes. This covalent immobilization of enzymes containing thiol is also useful for storing and protecting enzymes by immobilizing on thiopropyl resin.
The resin operates on covalent chromatography, forming mixed disulfides with thiol-containing proteins. By exchanging the labile thiopyridyl group, the resin effectively captures proteins with reversible cysteine thiol modifications.
Thiopropyl resin contains an active group of 2-pyridyl disulfide, with a concentration of 18-31 µmole 2-pyridyl disulfide/ml drained gel and a coupling capacity of around 10-20 mg protein/ml of drained gel. Since the matrix comprises 6% cross-linked agarose, it is chemically stable to commonly used aqueous buffers and additives, excluding azides.
There are several reasons why researchers prefer thiopropyl resin in proteomic applications.
- It provides a more straightforward workflow with increased specificity.
- It enables the selective enrichment and analysis of S-nitrosylation and reversible cysteine modifications in proteins.
- The resin-assisted approach allows for quantitative site-specific analysis of cysteine-based reversible modifications through liquid chromatography-tandem mass spectrometry (LC-MS/MS).
What is the mechanism of action of thiopropyl resin in protein purification?
Thiopropyl resin works by reversibly immobilizing thiol-containing molecules on the resin, resulting in mixed disulfides. As the sample passes through the resin, it interacts with the thiol-containing biomolecules and releases 2-thiopyridone. A reducing agent such as DTT or 2-mercaptoethanol can elute the bound molecules.
How is thiopropyl resin prepared for protein purification?
Commercially available resins are supplied in either dry forms or fully hydrated resin in suspension form. G-Bioscience offers thiopropayl resin in suspension form. Thiopropyl resin is typically supplied as a 50% slurry in 20% ethanol containing 1 mM EDTA. To ensure the resin's best performance and shelf life, store it at 4°C and protect it from light. Also, avoid using azides.
Prepare the resin. Pour an appropriate amount of resin suspension into a chromatography column. G-Bioscience offers a wide variety of empty chromatography columns. Allow the resin suspension to settle down in the column. If the resin is dry, you must perform the resin hydration step as recommended by the suppliers. For example, transfer an appropriate amount of thiopropyl resin to a beaker or tube and add water. Rehydrate the resin for 15 minutes at room temperature. Resuspend the resin well using a pipette tip and leave the tubes for another 15 minutes at room temperature. After removing and discarding the upper portion of the water, resuspend the settled resin and transfer the suspension to a chromatography column for further processing.
Wash and equilibrate the resin. To remove the storage buffer, wash the resin multiple times with water, phosphate buffer, or any appropriate coupling buffer without containing a reducing agent. To minimize thiol oxidation, degas the coupling or washing buffer by sonication for 20 minutes before starting the sample loading step.
Elution and protein capture. After washing, load the protein sample for immobilization or capture of thiol-containing proteins in the sample. After loading the protein sample, wash the column at least 5-bed volumes with the wash buffer to remove unbound proteins. Elution buffers containing reducing agents like DTT or 2-mercaptoethanol are used to release and elute captured proteins from the resin effectively.
Optimize and validate results. A preliminary titration study before the main experiment is recommended to determine optimal binding conditions. Likewise, wash the column with specific buffers before elution to ensure proper removal of 2-dipyridyl groups after protein binding.
Denaturing agents like SDS, urea, or guanidine HCl can also effectively expose all thiol groups and make them accessible for the reaction.
What factors should be considered when choosing thiopropyl resin for protein purification?
- Target protein characteristics. For optimal binding and elution, consider the target protein's size, charge, and hydrophobicity. G-Biosciences offers a wide variety of chromatographic purification resources.
- Sample characteristics. Know the characteristics of the sample and its impurities to help you choose a resin that can effectively separate the target protein from the sample matrix.
- Purification goal. Determine the desired outcome of the purification process to select the most suitable resin and method.
- Liquid chromatography technique. Choose the appropriate liquid chromatography technique based on the target protein's characteristics and the desired purification goal.
- Resin characteristics. When determining compatibility with the sample and purification requirements, consider the resin's active group, concentration, spacer, coupling capacity, pH stability, chemical stability, and physical stability.
- Stability and compatibility. To decide whether the resin suits the intended experiment, consider the purification process's requirements (temperature, pH, and reagents used).
Example Case Study: Here is an example of Sulfhydryl or thiol group-containing protein purification from fresh spinach leaves. 1.0-gram fresh spinach leaves were ground and homogenized in 3.0 ml PBS buffer. The homogenate was centrifuged at 10,000 x g for 15 minutes to obtain a clean lysate. After centrifugation, 3.0 ml of clear supernatant was collected.
G-Biosciences thiopropyl 1.0ml resin was packed in a 3.0 ml gravity flow column. The thiopropyl-packed resin was washed with 10.0ml PBS buffer by gravity flow method. A 3.0ml extract was applied to the isopropyl-packed resin column. The extract was allowed to pass through the column under gravity. After the extract passed through the packed resin column, the column was washed with 10.0ml PBS.
For the elution of thiol-containing proteins immobilized on the column, PBS containing 20.0 mM DTT was used. 10.0ml elution buffer was applied to the column, and the flow through was collected as a 1.0ml fraction. Ten fractions were collected, each fraction 1.0 ml. Eluted fractions were analyzed by scanning each fraction between 200nm and 500nm. Figure -1 shows scanning results from 1-4 fractions. Two peaks were observed, the first at 273nm and the second at 343nm. The first peak represented characteristic protein, whereas the second peak at 343nm represented 2-dipyridyl as it dissociated from the resin when disulfide-containing protein bonded with the resin eluted from the resin.
Protein analysis was performed on fractions 1-4 by denaturing gel electrophoresis (Figure 2). Maximum thiol-containing protein elution was observed in fractions 2 and 3. Two proteins with molecular weights 20kD and around 10kD were the dominant proteins in the elution fraction. There were several minor proteins.
This method shows rapid binding of thiol-containing proteins on the resin and easy and rapid elution of the captured protein with a reducing agent.
Figure 3 Protein Purification Supplies & Resources
Figure 3 Protein Purification Supplies & Resources
