Feb 25, 2026

Activity Assay V.2

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvodydzg4o/v2
  • A. Felicia Adebanjo1,
  • Madeline Walsh1,
  • Jessica Simon1,
  • Raining Wang1,
  • Melinda Wheelock1,
  • Aman Shihora1,
  • Sriram Pendyala1,
  • Allyssa Vandi1,
  • Dan Holmes1,
  • Douglas M. Fowler1,
  • Dustin J. Maly1
  • 1University of Washington
Melinda K. Wheelock
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvodydzg4o/v2
Protocol Citation: A. Felicia Adebanjo, Madeline Walsh, Jessica Simon, Raining Wang, Melinda Wheelock, Aman Shihora, Sriram Pendyala, Allyssa Vandi, Dan Holmes, Douglas M. Fowler, Dustin J. Maly 2026. Activity Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvodydzg4o/v2Version created by Melinda K. Wheelock
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: February 24, 2026
Last Modified: February 25, 2026
Protocol  Integer ID: 243955
Keywords: Transduction, Lentivirus, ERK inhibitor, activity assay with e40, description of activity assay, pem1 enrichments this protocol, pem1 enrichment, activity assay, e40, assay, intracellular braf variant activity, intracellular braf, erk2 phosphorylation reporter, activity assay this sop, seq mapk, signaling activity, variant activity, tdmcp
Abstract
This SOP describes the procedure for performing the LABEL-seq MAPK signaling activity assay using a tdMCP-Erk2 phosphorylation reporter to quantify intracellular BRaf variant activity in a pooled format.
Guidelines
Use BSL-2 practices when handling HEK293 cells.
Wear appropriate PPE
Handle TRIzol in chemical fume hood.
Materials
  • Tris BaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #252859
  • GlycerolMerck MilliporeSigma (Sigma-Aldrich)Catalog #G5516
  • Magnesium chloride hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #442611
  • Sodium fluorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S7920
  • PMSFMerck MilliporeSigma (Sigma-Aldrich)Catalog #P7626
  • RiboLock RNase Inhibitor (40 U/µL)Thermo FisherCatalog #EO0382
  • NaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9625
  • Doxycycline hyclateMerck MilliporeSigma (Sigma-Aldrich)Catalog #D5207
  • DMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
  • SCH772984MedChemExpressCatalog #HY-50846
  • DMEM, high glucoseThermo FisherCatalog #11965118
  • Fetal Bovine Serum, Tet system approved, USDA-approved regionsThermo Fisher ScientificCatalog #A4736401
  • Penicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122
  • Tergitol (NP-40 solution)Merck MilliporeSigma (Sigma-Aldrich)Catalog #NP40S

Cell lines

  • Library recombined in RPLP cell line
  • Activity standards cell stock
  • HEK293T cells for transduction

Plasmids

  • (pLJM1) 3xFlag-tdMCP-ERK2 K54R (kinase dead)-IRES-mTagBFP2
  • (pMDLg/pRRE) Gag and Pol packaging plasmid
  • (pRSV-REV) Rev packaging plasmid
  • VSV-G envelope plasmid
  • PEI

Drug Stocks

  • 20,000x Dox (90 millimolar (mM) ) - 40 µL Dox in DMSO
  • 20,000x SCH772984 (2 millimolar (mM) ) - 1.1753 µL SCH772984 in DMSO *Shortened to SCH in protocol

Medias

  • +Dox, +SCH772984 media
○ Dulbecco’s Modified Eagle Medium (1x) (DMEM) ○ 10% Fetal Bovine Serum (FBS) ○ 1x SCH772984 (100nM) ○ 1x Dox (2 µL )
  • Dox-free, +SCH772984 media
○ Dulbecco’s Modified Eagle Medium (1x) (DMEM) ○ 10% Fetal Bovine Serum (FBS) ○ 1x SCH772984 (100nM)

Buffers

  • Resuspension buffer

AB
Tris pH 7.650 mM
NaCl150 mM
Glycerol5%
MgCl24 mM
NaF10 mM
PMSF2 mM
Mini Halt protease inhibitor1 tablet/10mL
Phosphatase inhibitor cocktail 210μL/mL
Phosphatase inhibitor cocktail 310μL/mL
Ribolock RNase inhibitor4μL/mL
dummy RNA300nM
  • Lysis buffer

AB
Tris pH 7.650 mM
NaCl150 mM
Glycerol5%
MgCl24 mM
NaF10 mM
PMSF2 mM
Mini Halt protease inhibitor1 tablet/10mL
Phosphatase inhibitor cocktail 210μL/mL
Phosphatase inhibitor cocktail 310μL/mL
Ribolock RNase inhibitor4μL/mL
NP400.2%
Wash buffer

AB
Tris pH 7.650 mM
NaCl150 mM
Glycerol5%
MgCl24 mM
NaF10 mM
PMSF2 mM
NP400.1%

Beads

  • Streptavidin-conjugated agarose bead slurry (ThermoFisher Scientific, Catalog # SA10004)
Streptavidin AgaroseThermo FisherCatalog #SA10004
  • E40 DARPin stock
  • pEM1 DARPin stock

Buffer Recipes

For each LABEL-seq assay, you will need roughly the following volumes of buffers per 10M cells (= 1 mL lysate):
  • Resuspension buffer - 0.5 mL
  • Lysis buffer - 0.5 mL
  • Wash buffer - 0 mL L for abundance assay, 4 mL for activity assay

Final concentrations of buffers:

  • Resuspension buffer

○ 50 millimolar (mM) Tris pH 7.6 ○ 150 millimolar (mM) NaCl ○ 5% glycerol ○ 4 millimolar (mM) MgCl2 ○ 10 millimolar (mM) NaF ○ 2 millimolar (mM) PMSF ○ For abundance assay:
  • 1x Promega protease inhibitor
○ For activity assay:
  • 1 tablet/10mL mini Halt protease inhibitor
  • 10 µL phosphatase inhibitor cocktail 2
  • 10 µL phosphatase inhibitor cocktail 3
○ 4 µL Ribolock RNase inhibitor ○ 0 nanomolar (nM) dummy RNA

  • Lysis buffer

○ 50 millimolar (mM) Tris pH 7.6 ○ 150 millimolar (mM) NaCl ○ 5% glycerol ○ 4 millimolar (mM) MgCl2 ○ 10 millimolar (mM) NaF ○ 2 millimolar (mM) PMSF ○ For abundance assay:
  • 1x Promega protease inhibitor
○ For activity assay:
  • 1 tablet/10mL mini Halt protease inhibitor
  • 10 µL phosphatase inhibitor cocktail 2
  • 10 µL phosphatase inhibitor cocktail 3
○ 4 µL Ribolock RNase inhibitor ○ 0.2% NP40

  • Wash buffer

○ 50 millimolar (mM) Tris pH 7.6 ○ 150 millimolar (mM) NaCl ○ 5% glycerol ○ 4 millimolar (mM) MgCl2 ○ 10 millimolar (mM) NaF ○ 2 millimolar (mM) PMSF ○ 0.1% NP40

Stock buffers/reagents (prepare in advance)

5x RIPA (200mL)

Note
Make large stock, can be stored at room temperature

  • 6.057 g Tris
  • 8.677 g NaCl
  • 50 mL glycerol
  • 813 mg magnesium chloride hexahydrate
  • MillQ water to 200 mL
Adjust pH to 7.6 .

1M PMSF stock (1mL)

Note
Make large stock, can be stored at room temperature

  • 174 mg PMSF
  • 1 mL DMSO Sonicate to dissolve.

Buffers to make day of assay

Note
Ultimately, you only need the resuspension, lysis, and wash buffers which can each be made individually. For easier preparation, the following describes how to make each buffer from Buffer A (1x modRIPA without inhibitors) and Buffer B (1x modRIPA with inhibitors). Buffer A and Buffer B are used only to make resuspension, lysis, and wash buffers.

Buffer A - 1x modRIPA (10mL)

  • 2 mL 5x RIPA
  • 8 mL milliQ water
  • 4.2 mg NaF
  • 20 µL 1 Mass Percent PMSF stock (3.48 mg PMSF)
Sonicate to dissolve (can heat in water bath as needed, <5min). PMSF may crash out after adding to buffer, so make sure to fully redissolve.

Buffer B - modRIPA with inhibitors (10mL)

  • 10 mL Buffer A
  • 40 µL Ribolock RNase inhibitor
  • For abundance assay:
○ 200 µL 50x Promega protease inhibitor
  • For activity assay:
○ 1 mini Halt protease inhibitor tablet ○ 100 µL phosphatase inhibitor cocktail 2 ○ 100 µL phosphatase inhibitor cocktail 3

The mini Halt protease inhibitor tablet is the most difficult reagent to fully dissolve in solution, so add to Buffer A before adding anything else and sonicate to dissolve (can heat in water bath as needed, <5min).

Resuspension Buffer (1mL)

  • 1 mL Buffer B
  • 30 µL 10 micromolar (µM) dummy RNA

Lysis Buffer (1mL)

  • 1 mL Buffer B
  • 2 µL NP40
Sonicate to dissolve NP40 in solution. NP40 is a detergent so avoid vortexing.

Wash Buffer (1mL)

  • 1 mL Buffer A
  • 1 µL NP40
Sonicate to dissolve NP40 in solution. NP40 is a detergent so avoid vortexing.
Day Prior: pErk Bead Preparation
Combine the following:
350 µL Protein A bead slurry
350 µL tris-buffered saline
70 µL anti-pErk antibody
Rotate overnight at 4°C
Cell Transfection (48 hours before lysis)Untitled section
For each 15-cm dish, transiently transfect cells with pErk reporter:
Combine the following and incubate at room temperature for 15 minutes
17 µg 3xFlag-tdMCP-Erk2 reporter plasmid
1.7 µg pMAX-GFP
70 uL Fugene HD
2 mL serum-free DMEM
Add mixture drop-wise to cells.
Maintain cells in 2ug/mL doxycycline and 100nM SCH772984
Incubate cells for 48 hours

Remove SCH772984 from cell media 24 hours prior to cell lysis.
Cell Harvest and Lysis
Remove media and rinse cells with warm DPBS.
Trypsinize and collect cells into 15 mL tubes.
Spin down cells at 800 x g for 5 min. Remove media.
Resuspend each 15 cm plate (approx 20 M cells) of cells in 1.0 mL cold Resuspension Buffer.
To the resuspended cells, immediately add an equal volume (1 mL per 20 M cells) cold Lysis Buffer containing 0.2% NP-40 to lyse cells. Mix by pipetting.
Keep on ice; this marks the start of lysis.
Centrifuge at 17,000 × g for 10 min at 4°C.
Lysate Division and Bead Incubation
Pool lysates for each replicate if applicable.
Per 15 cm plate, split into:
1.75 mL for pErk IP
250 µL for Flag IP
Add beads:
Per 1.75 mL, add 175 µL pErk beads (previously washed 2x with wash buffer and resuspended in same volume)
Per 250 µL, add 25 µL anti-Flag beads (previously washed 2x with wash buffer and resuspended in same volume)
Rotate end-over-end for 2 hours at 4°C.
Bead Washing and RNA Extraction
Wash each bead sample 3X with 1 mL cold Wash Buffer.
Remove all buffer.
Resuspend beads in:
75 µL nuclease-free water
300 µL TRIzol
Store at −20°C or proceed to RNA extraction.
RNA extraction & cleanup
Extract RNA according to TRI-Isolate kit instructions.
Clean and concentrate RNA using Zymo RNA Clean & Concentrator kit; elute in ~15 µL nuclease-free water.
Reverse Transcription & PCR AmplificationUntitled section
Reverse transcription: use SuperScript IV with gene-specific RT primer JJS1 (TTACGACTCGTCGTCACT) following manufacturer protocol. Use RNase inhibitor throughout setup.
PCR round 1: amplify the circRNA barcode region using the round-1 forward primer (e.g., JJS8) and the appropriate round-1 reverse primer(s) matching your library build (JJS9–JJS15). Use high-fidelity polymerase (Q5). Cycle numbers: test to avoid over-amplification (e.g., 18–22 cycles; choose based on input).
PCR round 2 (adapter/indexing): perform a second PCR to append Illumina adapters and indices using the round-2 forward primers (JJS16–JJS23, choose one per sample) and the round-2 reverse primer JJS24; add index primers JJS27/JJS28 as required. Purify PCR products (AMPure XP) and QC by gel / Bioanalyzer.
Quantify amplicons, pool equimolarly and sequence on Illumina platform (or send to Amplicon-EZ). Provide custom read primers JJS25/JJS26 to the sequencing provider if you are using non-standard read priming.
Sequence on Illumina NextSeq 2000 - use custom barcode read primers if required (JJS25/JJS26).
Protocol references
Simon JJ, Fowler DM, Maly DJ. Multiplexed profiling of intracellular protein abundance, activity, interactions, and druggability with LABEL-seq. Nat Methods. 2024 Nov;21(11):2094-2106. doi: 10.1038/s41592-024-02456-7. Epub 2024 Oct 21. PMID: 39433876; PMCID: PMC11785348.