Binding biotinylated nucleic acids, antibodies, or proteins to Hydrophilic Streptavidin Magnetic Beads (NEB #S1421) for Pull-Down experiments
Materials:
NEB Product | |
Hydrophilic Streptavidin Magnetic Beads | S1421S |
Magnetic rack sized appropriately for your experiment | |
96-well Microtiter Plate Magnetic Separation Rack | S1511S |
6-tube Magnetic Separation Rack for 1.5 mL microfuge tubes | S1506S |
12-tube Magnetic Separation Rack for 1.5 mL microfuge tubes | S1509S |
50 mL Magnetic Separation Rack for 50 mL conical tubes | S1507S |
Biotinylated bait (nucleic acid, antibody or protein) Binding/Wash buffer for nucleic acids: 10 mM Tris-HCl pH 7.5, 1 M NaCl, 1 mM EDTA Binding/Wash buffer for antibodies or proteins: 1X PBS pH 7.4 (+ 0.01% w/v BSA and/or 0.05% Tween-20 to reduce non-specific binding) Optional: NanoDrop/spectrophotometer |
Protocol:
- Vortex/mix the tube containing the Hydrophilic Streptavidin Magnetic Beads to fully resuspend them.
- Transfer the required volume of beads for your experiment to a sterile tube. Hydrophilic Streptavidin Magnetic Beads bind 400 pmol of 25 bp single stranded DNA (ssDNA) or 30 ug of biotinylated antibody or protein per milligram of beads; the beads are supplied at 4 mg/mL.
- Note: Optimal loading density of biotinylated bait on beads needs to be determined empirically. We recommend these values as a starting point.
- Place the tube in the magnetic rack and allow the beads to pellet; remove the storage buffer.
- Remove the tube from the magnetic rack. Equilibrate the beads with appropriate binding/wash buffer by mixing the beads thoroughly in a volume of binding buffer equal to or greater than the volume of beads transferred in step 2.
- Place the tube in the magnetic rack and allow the beads to pellet. Remove the binding/wash buffer.
- Repeat steps 4 and 5 twice more for a total of 3 equilibration washes.
- Resuspend the biotinylated bait in appropriate binding buffer (adjusting the NaCl concentration of this solution to be between 0.5 and 1 M after resuspension for biotinylated nucleic acid baits)
- Optional: Measure the A260 (nucleic acid) or A280 (antibody/protein) of the resuspended biotinylated bait to determine amount of target bound
- Add the resuspended biotinylated bait to pelleted and equilibrated magnetic beads. Mix thoroughly by pipet mixing or gentle vortexing.
- Incubate with mixing (rotisserie-style end-over-end mixing or ThermoMixer/equivalent) for > 30 minutes at room temperature or 4°C, depending on the stability of your biotinylated bait.
- Place the tube in the magnetic rack and allow the beads to pellet.
- Optional: Measure the A260 or A280 of the solution to quantify how much biotinylated bait remains. The difference between the first measurement and this measurement is a good approximation of the amount of biotinylated bait bound to the Streptavidin Magnetic Beads
- Wash the beads with the appropriate binding/washing buffer (or a wash buffer of higher stringency) as in steps 4-6.
- Resuspend the beads in a buffer compatible with your specific pull-down experiment; proceed directly to the pull-down experiment.