Labeling with SNAP-tag® Technology Troubleshooting Guide

Application
Problem Possible Cause Solution
Cellular Labeling No labeling Fusion protein
not expressed
  1. Verify transfection
  2. Check expression of fusion protein via Western blot or SDS-PAGE with Vista Green label
Weak labeling Poor expression and/or
insufficient exposure of
fusion protein to substrate
  1. Increase substrate concentration
  2. Increase incubation time
Rapid turnover of fusion protein
  1. Analyze samples immediately or fix cells directly after labeling
  2. Label at lower temperature (4°C or 16°C)
High background Non-specific binding of
substrates
  1. Reduce substrate concentration and/or incubation time
  2. Allow final wash step to proceed for up to 2 hours
  3. Include fetal calf serum or BSA during labeling
Signal strongly reduced after short time Instability of
fusion protein
  1. Fix cells
  2. Switch tag from N-terminus to C-terminus or vice versa
Photobleaching
  1. Add commercially available anti-fade reagent
  2. Reduce illumination time and/or intensity
Labeling in Solution Precipitation Insoluble fusion
  1. Test from pH 5.0 to 10.0
  2. Optimize salt concentration [50 to 250 mM]
  3. Add 0.05 to 0.1% Tween 20
Weak or no labeling Exhaustive labeling has not been achieved
  1. Increase incubation time to 2 hrs at 25°C or 24 hrs at 4°C
  2. Reduce the volume of protein solution labeled
  3. Check expression of fusion protein via SDS-PAGE with Vista Green label
Loss of activity Instability of
fusion protein
  1. Reduce labeling time
  2. Decrease labeling temperature (4°C or 16°C)