Labeling Mammalian Cell Lysates (S9147)
Dissolve one vial of SNAP-tag substrate (50 nmol) in 50 μl of DMSO to yield a labelling stock solution of 1 mM SNAP-tag substrate. Mix by vortexing for 10 minutes until all the SNAP-tag substrate is dissolved. Store this stock solution in the dark at 4°C, or for extended storage at -20°C. Different stock concentrations can be made depending on your requirements. The substrate is soluble up to at least 10 mM.
- Harvest cells by trypsinization following established protocols.
- Wash cells twice with PBS.
- Lyse cells by suspending in reaction buffer at 104–105 cells per 20 µl. Reaction buffer is 50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 0.1% Tween-20, 1 mM DTT and an EDTA-free protease inhibitor cocktail (e.g., Complete™, Roche).
- Dilute the 1mM SNAP-Vista Green stock solution 1:4 in fresh DMSO to yield a 250μM stock solution. Add 2 µl of the SNAP-Vista Green 250μM stock solution to 18 µl of cell lysate. Mix well by pipetting up and down several times.
- Incubate in the dark for 30 minutes at room temperature.
- Add an appropriate volume of concentrated SDS-PAGE sample buffer and proceed with sample preparation and SDS-PAGE according to the gel manufacturer’s instructions.
- After the gel is run, immediately obtain a fluorescent image using a laser scanner with 488 nm excitation or a UV transilluminator and an appropriate camera (Polaroid or digital). Excitation at 488 nm will give the best results. The fluorescence is an intense green.
- After fluorescent imaging, standard fixing and staining protocols can be used to detect the non-fluorescent proteins.
- Dissolve the vial of SNAP-Vista Green (50 nmol in 50 μL of fresh DMSO to yield a labeling stock solution of 1 mM SNAP-tag substrate. Mix by vortexing for 10 minutes until all the SNAP-tag substrate is dissolved. Dilute this 1 mM stock solution 1:4 in fresh DMSO to yield a 250 μM stock for labeling proteins in vitro.
- Set up the reactions, in order, as follows:
42 μl 1X 50 mM Dtt 1 μl 1 mM 50 μM SNAP-tag
5 μl 5 μM 250 μM SNAP-tag
2 μl 10 μM Total Volume 50 μl
- Incubate in the dark for 30 minute at 37°C.
- Run sample on an SDS-PAGE gel and detect using a fluorescent gel scanner or store samples at -20°C or -80°C in the dark.
Removal of Unreacted Substrate (optional)
After the labeling reaction the unreacted substrate can be separated from the labeled SNAP-tag fusion protein by gel filtration or dialysis. Please refer to the vendor's instructions for the separation tools you are using.
Notes for Labeling in vitro
We recommend the routine addition of 1 mM DTT to all buffers used for handling, labeling and storage of the SNAP-tag. The stability of the SNAPtag is improved in the presence of reducing agents; however it can also be labeled in their absence. If handling at temperatures above 4°C is minimized.
SNAP-tag fusion proteins can be purified before labeling, but the labeling reaction also works in non-purified protein solutions (including cell lystates).
Troubleshooting for Labeling in vitro Solubility
If solubility problems occur with your SNAP-tag fusion protein, we recommend testing a range of pH (pH 5.0-pH 10.0) and ionic strengths. The salt concentration may also need to be optimized for your particular fusion protein (50-250 mM).
Loss of Protein Due to Aggregation or Sticking to Tube
If stickiness of the fusion protein is a problem we recommend adding Tween 20 at a final concentration of 0.05% to 0.1%. The SNAP-tag activity is not affected by this concentration of Tween 20.
If exhaustive labeling of a protein sample is not achieved using the recommended conditions, try the following protocol modifications: Increase the incubation time to two hours total at 25°C or to 24 hours at 4°C; or halve the volume of protein solution labeled. Both approaches may be combined.
If the SNAP-tag fusion has been stored in the absence of DTT or other reducing agent, or has been stored at 4°C for a prolonged period, its activity may be compromised. Include 1 mM DTT in all solutions of the SNAP-tag fusion protein, and store the fusion protein at -20°C.
Using less than the recommended amount of substrate stock solution can significantly slow down the reaction rate.
Loss of Activity of Protein of Interest
If your fusion protein is particularly sensitive to degradation or to loss of activity, you can try reducing the labeling time or decreasing the labeling temperature. If you label at 4°C we recommend overnight incubation.
Notice to Buyer/User: The Buyer/User has a non-exclusive license to use this system or any component thereof for RESEARCH AND DEVELOPMENT PURPOSES ONLY. Commercial use of this system or any components thereof requires a license from New England Biolabs, Inc., 240 Count Road, Ipswich, MA 01938.