Low throughput protocol for immunoprecipitation followed by mass spectrometry of cells stably expressing an HA-tagged protein
Harper JW
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Abstract
Analysis of protein complexes by mass spectrometry provides a powerful approach for identifying proteins that associate with other proteins. Frequently, this can be done by expressing the protein of interest with an epitope tag, such as a Hemagglutinin-A (HA) epitope, using either a stably expressed lentivirus or by gene editing the HA epitope into the gene of interest. The protocol has been used extensively to create the Bioplex protein interaction network [Huttlin et al Nature. 545:505-509 (2017); Huttlin et al Cell, 162: 425-440 (2015)].
Before start
This protocol is for 293T cells but has been used broadly for many cell types. Typically, cells used have the proteins to be immunoprecipitated have stably expressed HA-tagged proteins via lentivirus vectors, or proteins fused with an HA epitope using gene editing.
Cell harvest:
- 2 x 15cm plate or 5 x 10cm plates per IP.
- Include HA-tagged GFP bait as a control.
Attachments
Steps
Cell Harvest
Wash plates 2x with cold PBS, then add 5mL per plate.
Gently pipette up and down to dislodge cells and homogenize or gently scrape.
Transfer to 15 mL conical tube and pellet cells 3000rpm, discard sup.
Add 1mL and transfer to 2 mL tube.
Spin, aspirate PBS, and snap freeze pellet.
Store at -80°C or use immediately.
Quick thaw frozen 293T pellets in 2mL tubes in 37°C water bath for approximately 0h 0m 3s.
Transfer to ice metal block and add 1.2mL.
Once pellet is thawed, pipette up and down to resuspend and tumble tubes for 0h 20m 0s in the cold room at 4°C to lyse cells.
Spin at 16.1rcf in pre-chilled bench-top centrifuges to clear lysate.
Reserve 25µL -50µL for QC protein assay and western blot of input, if desired.
Carefully transfer remaining supernatant to fresh 1.5 mL tube containing 40µL.
Incubate cleared lysate with beads for 3h 0m 0s with tumbling in the cold room at 4°C.
Spin samples 3000rpm,4°C to pellet beads in centrifuge.
Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!)
Add 1mL to each tube, and gently resuspend beads by shaking.
Repeat spin/wash step 3 more times for a total of 4 x 1 mL washes with detergent present:
(Wash 1/3): Spin samples 3000rpm,4°C to pellet beads in centrifuge. Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!). Add 1mL to each tube, and gently resuspend beads by shaking.
(Wash 2/3): Spin samples 3000rpm,4°C to pellet beads in centrifuge. Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!). Add 1mL to each tube, and gently resuspend beads by shaking.
(Wash 3/3): Spin samples 3000rpm,4°C to pellet beads in centrifuge. Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!). Add 1mL to each tube, and gently resuspend beads by shaking.
Perform 3 x 1 mL washes in the absence of detergent 50mM Tris/150mM NaCl, without NP-40:
(Wash 1/3): Spin samples 3000rpm,4°C to pellet beads in centrifuge. Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!). Add 1mL to each tube, and gently resuspend beads by shaking.
(Wash 2/3): Spin samples 3000rpm,4°C to pellet beads in centrifuge. Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!). Add 1mL to each tube, and gently resuspend beads by shaking.
(Wash 3/3): Spin samples 3000rpm,4°C to pellet beads in centrifuge. Carefully decant supernatant using aspirator or 1mL pipette (be careful not to aspirate beads!). Add 1mL to each tube, and gently resuspend beads by shaking.
Carefully aspirate remaining wash buffer. Use gel loading tip and pipettor to remove as close to beads as possible.
Add 100µL (HA peptide elution buffer + 250µg/mL).
Incubate in shaker at 37°C (gentle shaking) for 0h 30m 0s.
Collect bead eluate by centrifuging 3000rpm.
Repeat elution with equal volume of HA peptide; incubate second elution for 15 min:
Add 100µL (HA peptide elution buffer + 250µg/mL).
Incubate in shaker at 37°C (gentle shaking) for 0h 15m 0s.
Collect bead eluate by centrifuging 3000rpm.
Transfer eluate to labeled 1.5mL tubes; freeze at -80°C.
Proceed to TCA precipitation.
TCA Precipitation
The following steps constitute the TCA precipitation/acetone wash and trypsinization in preparation for analysis by mass spectrometry: Can perform TCA precipitation at 4°C or for 0h 45m 0s On ice.
Add 55µL to samples (assuming 2 x 100µL), vortex to mix, then
gently spin to ensure TCA is not in tube caps.
Spin max speed 13000rpm,4°C; carefully aspirate all but 30µL.
Wash pellet with 1mL cold 10% made in HPLC grade water.
Spin max speed 0h 15m 0s, vacuum as in Step 2.
Wash with 1mL.
Spin max speed 0h 10m 0s, vacuum.
Repeat Acetone wash 2 more times (3 acetone washes total):
(Wash 1/2): Wash with 1mL. Spin max speed 0h 10m 0s, vacuum.
(Wash 2/2): Wash with 1mL. Spin max speed 0h 10m 0s, vacuum.
Air dry or use speedvac to dry pellet for digest, must be completely dry, as acetone can cause peptide modifications.
Trypsin Digestion
Resuspend dried pellet in 40µL (digest buffer).
Spot check with 0.2µL for a couple of samples to ensure 8.5.
Add 100ng (Thermo). Stock is 20ng in 20µL (measure to confirm 20L for each tube). This is 1µg/µL, make a master mix of trypsin digest buffer and add 40µL to each sample.
Incubate at 37°C for 6h 0m 0s (warm room or thermomixer, can shake gently).
Acidify with 2 digest volumes of 5% in HPLC grade water. (For 40L digest, add 80µL.)
Spot check pH for a couple of samples to ensure 2.
Proceed to stage tip followed by analysis by mass spectrometry.
