Use SNAP-Capture Pull Down Resin (S9144)
Overview
This protocol describes the use of SNAP-Capture Magnetic Beads in small scale batch format. It can be scaled-up to fit the requirements for specific applications.
Protocol
Equilibration of Resin
The SNAP-Capture Pull Down Resins are stored in 50% isopropyl alcohol. The storage buffer must be exchanged with immobilization buffer before use.
- Carefully and thoroughly resuspend the 25% SNAP-Capture Pull Down Resin suspension. Immediately withdraw an 80 µl sample to a 1.5 ml microcentrifuge tube. The 80 µl sample contains 20 µl bed-volume resin.
- Add 1 ml of immobilization buffer to the resin. Vortex gently to mix. Centrifuge for 1 minute at 11000 x g. Carefully remove and discard the supernatant, resuspend and repeat 2 times.
Immobilization of SNAP-tag Fusion Protein
Prepare a protein solution containing up to 1 mg/ml SNAP-tag fusion protein to be immobilized in an appropriate buffer containing at least 1 mM DTT. We recommend the use of at least 100 µl of this solution for each immobilization reaction.
- Add this protein solution to the SNAP-Capture Pull Down Resin in a 1.5 ml microcentrifuge tube (20 µl bed-volume resin) prepared as above.
- Incubate with mixing for 1 hour at room temperature. Alternatively, incubate overnight at 4°C with mixing.
Washing Step
The washing step removes non-specifically bound protein after the immobilization reaction.
- Wash the resin at least three times with 1 ml of immobilization buffer. To wash, add immobilization buffer to the resin and agitate for one minute, centrifuge (1 minute at 11,000 x g at 4°C), carefully remove and discard the supernatant, and repeat.
- The SNAP-Capture Pull Down Resin immobilized fusion protein is now ready for further use (e.g. pull down assay).
Troubleshooting
If sufficient immobilization of a protein sample is not achieved using the recommended conditions, try the following protocol modifications: Double the incubation time to two hours total at 25°C or to 24 hours at 4°C. If you still have poor immobilization results, we recommend checking the activity of the SNAP-tag (see below).
If your fusion protein is particularly sensitive to degradation or to loss of activity, you can try reducing the immobilization time or decreasing the immobilization temperature. If you immobilize at 4°C we recommend overnight incubation. Addition of a standard EDTA-free protease inhibitor cocktail, although not generally necessary for the SNAP-tag itself, may also help.
The activity of the SNAP-tag may have been partially or completely lost. This may be due to extended storage of non-reacted SNAP-tag fusion proteins at 4°C or above. The sensitivity of the SNAP-tag to inactivation is decreased if a reducing agent such as 1 mM DTT is added.
If the activity of the SNAP-tag is affected, we recommend analyzing a small fraction of it on a PAGE gel using SNAP-Vista Green (NEB# S9147 ) to confirm that the SNAP-tag is active.
If you encounter problems with the activity we recommend thawing another sample of your protein or reexpressing and repurifying the SNAP-tag fusion protein following the advice given in the SNAP-tag plasmid instructions.
Washing Step
It is important to maintain the functional integrity of the SNAP-tag fusion protein during the washing step. Although the SNAP-tag is covalently linked to SNAP-Capture Pull Down Resin, and will remain bound under harsh conditions, we recommend washing the resin before use only under mild buffer conditions to minimize the possible loss of fusion protein function. Fusion protein stability is highly protein dependent.
Some partially purified fusion proteins may exist as multimers and higher molecular weight aggregates. It may not be possible to wash aggregates away under mild conditions. This would not interfere with most applications but these proteins could leach off the resin under extreme denaturing conditions (e.g. SDS).
Protein Interaction Assay
Depending on the strength and properties of the particular interacting proteins in a protein interaction study, the following post-interaction washing parameters can be varied to improve the specificity of removing weakly interacting proteins; salt concentration (100–500 mM), non-ionic detergents (0.1–1%), glycerol (10–30%), DTT (0.1–1 mM) or EDTA (1–10 mM).
Addition of a standard protease inhibitor cocktail, although not generally necessary for the SNAP-tag itself, may be useful to prevent degradation of the interacting proteins when working with complex mixtures such as lysate.
