Biotinylation by antibody recognition V1.0
Biotinylation by antibody recognition - A novel method for proximity labeling
The goal of this protocol is to identify by proteins in proximity to an antigen of interest. The antigen is identified by a primary antibody. A secondary HRP-conjugated antibody drives the deposition of phenol-biotin on adjacent proteins. These are extracted and analysed by mass-spectrometry.
PSBT - PBS with 0.1% tween 20. Make fresh: formaldehyde, BSA, sodium ascorbate and sodium deoxycholate.
Use 2M HeLa cells or mass equivalent (~7mg) of any other cell type.
1. Fix sample with 4% formaldehyde in PBST for 10min at RT.
2. Wash twice with PBST.
3. For SILAC: Calculate cell number or protein concentration and mix equal amounts into a single tube.
4. Adjust volume to 1ml and permeabilize for 7min in PBS with 0.5% triton X-100.
5. Wash with PBST.
6. Add H2O2 to 0.5% for 10m, wash twice with PBST.
7. Block with 1% BSA in PBST for 2h, shaking.
8. Stain with MAB3211 (1:500) antibody over-night at 4C.
9. Wash samples over 1h in PBST with at least 3 buffer changes.
10. Add Secondary HRP antibody at 1:1000 in 1% BSA for 1h.
11. Wash samples over 2h in PBST with at least 3 buffer changes.
12. Pre-incubate with 3ul TSA (Tyramide Signal Amplification) reagent for 5 min. Replace buffer with 150ul reaction buffer and incubate for 1-7m (longer incubation will result in stronger signal, but also more non-specific binders). Stop reaction with 1ml 500 mM sodium ascorbate.
13. Wash three times with PBST.
14. Validate labeling by immunofluorescence of subsamples. Add a red secondary fluorescent ab, DAPI, and FITC-streptavidin and incubate for 30m at RT. Wash samples over 1h in PBST with at least 3 buffer changes. Mount subsamples on slides with vectashield. Labeling worked if strong green (biotin) signal is seen co-localizing with the red signal from the secondary fluorescent antibody.
15. Adjust volume to 0.1ml PBST, add 30ul of 10% SDS and 20ul of sodium deoxycholate 10%, heated for 99C for 1h with mild shaking.
16. Centrifuge at max speed for 5m.
17. Adjust supernatant volume to 1ml with PBST and add 250ul of pre-washed streptavidin beads. Incubate for 2h at RT. Keep unbound. For beads: wash once with PBST, twice with PBST with 1M NaCl, and twice more with PBS (*NOT PBST*).
18. Move beads to new tube. Reduce volume to 100ul, subsamples (~10%) can be used for Western validations. For westerns with streptavidin-HRP, a very strong signal, either of multiple bands or of a smear, is expected.
19. Precipitate unbound proteins by adding 6 volumes of cold acetone with 10mM NaCl and placing in -20C for 1h, followed by centrifugation at 14,000 rpm for 15m.
20. Resuspend beads/pellet in 90 µl 50 mM ABC (ammonium bicarbonate) and add 2 µl 500 mM DTT. Incubate beads for 30m at 37C with mixing, unbound for 30m at 56C.
21. Spike all samples with 10 µl 550 mM Iodoacetamide and leave a further 20 mins at RT, with mixing and in the dark.
22. Pellet beads and wash twice with 200 µl 50 mM ABC. Spike unbound with 20 µl 100 mM DTT.
23. Resuspend beads in 200 µl of 50 mM ABC containing 2 µg of trypsin and incubate at 37°C with mixing. Add 2 µg of trypsin to unbound.
24. Add an additional 1 µg of trypsin to each sample and incubate for a further 2 hours at 37°C.
25. Pellet the beads at low speed and transfer the supernatant to a fresh tube.
26. Rinse the beads with 150 µl of HPLC-grade H20 and combine with the rest of the supernatant.
27. Rinse the beads with 150 µl 1% trifluoroacetic acid (in H2O) and combine with the rest of the supernatant. Add 5 µl 100% trifluoroacetic acid to the unbound.
28. Spin the pooled eluant at 14,000 rpm for 2 minutes and transfer supernatant to a fresh tube.
29. Reduce sample volume in the vacuum centrifuge, Zip Tip.
Send for mass spec or freeze.
Daniel.bar at mail.huji.ac.il