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#314 Pharmacology Complete

IC50: ERK1 kinase + inhibitor X (ADP-Glo, 10 pt)

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IC50 dose-response ERK1-kinase ADP-Glo lead-optimization

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This template captures a 10-point dose-response IC50 determination of inhibitor X against recombinant ERK1 (MAPK3) kinase using the Promega ADP-Glo luminescent kinase assay in 384-well format, performed for lead-optimization compound profiling.

Goal: determine inhibitor X potency (IC50) against ERK1 with a Hill slope and Z’-factor confirming clean assay behavior, supporting SAR analysis for the inhibitor series targeting the MAPK signaling pathway.

Method overview: 10-point 3-fold serial dilution of inhibitor X from 10 µM to 0.5 nM in DMSO, then diluted 100-fold into kinase reaction buffer for 1% final DMSO. Recombinant ERK1 at 5 nM final, MBP substrate at 1 µM final, ATP at 50 µM (apparent Km), 25 mM HEPES pH 7.4, 10 mM MgCl₂, 1 mM DTT, 0.01% Tween-20. 5 µL kinase + inhibitor pre-incubation (15 min RT), 5 µL substrate + ATP start, 60-minute reaction at RT. 10 µL ADP-Glo Reagent quench + 30-min ADP→ATP conversion, 20 µL Kinase Detection Reagent, 60-min luminescence development, read on Synergy Neo2 luminometer.

Expected: Inhibitor X IC50 of 280 ± 40 nM (consistent with prior ERK1 screening for this scaffold), Hill slope between 1.0 and 2.0 (single-site behavior), Z’-factor > 0.5 (assay window robust), R² > 0.99 for 4PL fit. Reference inhibitor (SCH772984) IC50 within 2-fold of published 5 nM (assay validity control).

Last saved: 34 seconds ago

13 fields loaded from IC50 ADP-Glo Kinase baseline (SOP v1.2)

Target

Target

text

ERK1 (MAPK3)

Target conc

number

5nM

Substrate

text

MBP (myelin basic protein)

ATP conc

number

50µM (Km app)

Compound & Dose

Compound

text

Inhibitor X (compound ID INH-2026-0341)

High dose

number

10µM

Dilution factor

number

3

Dose points

number

10

Replicates

number

4

Curve Fit & QC

IC50

number

280nM

Hill slope

number

1.4

Z′-factor

number

0.86

R²

number

0.997

compound_plate_map.pdf

198 KB

luminescence_raw.csv

24 KB

ic50_4PL_prism.xlsx

108 KB

01

Thaw compound stock + reference inhibitor SCH772984 from -80 °C

On dry ice, brief vortex

0:10

02

Prepare 10-point 3-fold dilution series in DMSO (10 mM → 0.5 µM source)

Source plate D1

0:15

03

Echo-dispense 50 nL per well of 384-well assay plate (4 reps × 10 doses + ctrls)

Echo 550 acoustic

0:08

04

Dispense 5 µL kinase mix (10 nM ERK1 in 1× buffer) per well; pre-incubate 15 min RT

0:15

05

Dispense 5 µL substrate + ATP mix (2 µM MBP + 100 µM ATP) per well; start kinase reaction

0:05

06

Incubate 60 min at RT (kinase reaction)

1:00

07

Dispense 10 µL ADP-Glo Reagent per well; incubate 40 min RT (ADP→ATP)

0:42

08

Dispense 20 µL Kinase Detection Reagent per well; incubate 60 min RT (luciferase)

1:02

09

Read luminescence on Synergy Neo2 (10 s integration per well); export raw CSV

BioTek run 2026-0604-1432

0:08

10

4PL fit in GraphPad Prism, extract IC50 + Hill + Z' + R²; archive workbook

IC50 280 nM · slope 1.4 · Z' 0.86 · R² 0.997

0:20

	A	B	C	D	E	F	G
1	Dose (nM)	Rep 1	Rep 2	Rep 3	Rep 4	Mean RLU	% Activity
2	10000	12,841	12,503	13,108	12,772	12,806	5
3	3333	15,124	14,891	15,308	15,142	15,116	9
4	1111	23,008	22,891	23,401	23,099	23,100	18
5	370	57,221	58,109	56,892	57,944	57,541	49
6	123	90,418	91,287	89,914	90,872	90,623	78
7	41	105,891	106,442	104,981	106,182	105,874	90
8	14	112,109	111,541	113,008	112,494	112,288	95
9	4.5	115,442	114,892	116,201	115,109	115,411	98
10	1.5	116,108	115,872	116,991	116,341	116,328	99
11	0.5	116,418	115,991	116,712	116,108	116,307	99
12	DMSO ctrl	117,108	116,872	117,341	116,994	117,079	100
13	no kinase	8,109	7,891	8,341	8,108	8,112	0

EXP-2026-0529 HTS hit triage — 200 cmpds @ 10 µM K. Patel May 29

EXP-2026-0610 IC50 — analogs 342-347 (SAR series) K. Patel Jun 10

SOP-0207 IC50 ADP-Glo Kinase SOP v1.2 QA Team Feb 18

KIT-0214 Promega ADP-Glo Kinase Assay insert Promega Docs Mar 12

TGT-ERK1 ERK1 (MAPK3) recombinant kinase datasheet Target DB Jan 8

Recombinant ERK1 (MAPK3), active

#PV3311 · Lot 2562842

In stock

MBP substrate (myelin basic protein)

#M1891 · Lot SLBS9421A

In stock

ATP (10 mM working stock)

in-house · Lot 2026-04

In stock

ADP-Glo Reagent

#V6930 · Lot 0000456871

In stock

Kinase Detection Reagent

#V6930 · Lot 0000456871

In stock

SCH772984 (reference ERK1/2 inhibitor)

#S7101 · Lot SLBT1284

Low

Compound source plate (DMSO master)

-80 °C · Compound Mgmt Freezer C-4 · Box CMP-2026-Q2

×1

ERK1 kinase aliquots (10 µL working)

-80 °C · Freezer A-1 · Drawer 2 · Box ENZ-KIN-2026

×24

ADP-Glo reagents (working aliquots)

-20 °C · Freezer A-1 · Drawer 3 · Box ADP-GLO-WK

×6

Visibility

Discovery BiologyK. Patel

Can write

K. PatelDr. R. Singh

Color

Stroke width

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{
    "experiment": {
        "id": 314,
        "custom_id": "IC50-ERK1-2026-007",
        "title": "IC50: ERK1 kinase + inhibitor X (ADP-Glo, 10 pt)",
        "template": "IC50 Dose-Response",
        "template_version": "v3.2",
        "category": "Pharmacology",
        "status": "Complete",
        "started_on": "2026-07-20",
        "tags": [
            "IC50",
            "dose-response",
            "ERK1-kinase",
            "ADP-Glo",
            "lead-optimization"
        ]
    },
    "main_text": "This template captures a 10-point dose-response IC50 determination of inhibitor X against recombinant ERK1 (MAPK3) kinase using the Promega ADP-Glo luminescent kinase assay in 384-well format, performed for lead-optimization compound profiling.\nGoal: determine inhibitor X potency (IC50) against ERK1 with a Hill slope and Z’-factor confirming clean assay behavior, supporting SAR analysis for the inhibitor series targeting the MAPK signaling pathway.\nMethod overview: 10-point 3-fold serial dilution of inhibitor X from 10 µM to 0.5 nM in DMSO, then diluted 100-fold into kinase reaction buffer for 1% final DMSO. Recombinant ERK1 at 5 nM final, MBP substrate at 1 µM final, ATP at 50 µM (apparent Km), 25 mM HEPES pH 7.4, 10 mM MgCl₂, 1 mM DTT, 0.01% Tween-20. 5 µL kinase + inhibitor pre-incubation (15 min RT), 5 µL substrate + ATP start, 60-minute reaction at RT. 10 µL ADP-Glo Reagent quench + 30-min ADP→ATP conversion, 20 µL Kinase Detection Reagent, 60-min luminescence development, read on Synergy Neo2 luminometer.\nExpected: Inhibitor X IC50 of 280 ± 40 nM (consistent with prior ERK1 screening for this scaffold), Hill slope between 1.0 and 2.0 (single-site behavior), Z’-factor > 0.5 (assay window robust), R² > 0.99 for 4PL fit. Reference inhibitor (SCH772984) IC50 within 2-fold of published 5 nM (assay validity control).",
    "extra_fields": {
        "Target": {
            "Target": {
                "type": "text",
                "value": "ERK1 (MAPK3)"
            },
            "Target conc": {
                "type": "number",
                "value": "5 nM"
            },
            "Substrate": {
                "type": "text",
                "value": "MBP (myelin basic protein)"
            },
            "ATP conc": {
                "type": "number",
                "value": "50 µM (Km app)"
            }
        },
        "Compound & Dose": {
            "Compound": {
                "type": "text",
                "value": "Inhibitor X (compound ID INH-2026-0341)"
            },
            "High dose": {
                "type": "number",
                "value": "10 µM"
            },
            "Dilution factor": {
                "type": "number",
                "value": "3"
            },
            "Dose points": {
                "type": "number",
                "value": "10"
            },
            "Replicates": {
                "type": "number",
                "value": "4"
            }
        },
        "Curve Fit & QC": {
            "IC50": {
                "type": "number",
                "value": "280 nM"
            },
            "Hill slope": {
                "type": "number",
                "value": "1.4"
            },
            "Z′-factor": {
                "type": "number",
                "value": "0.86"
            },
            "R²": {
                "type": "number",
                "value": "0.997"
            }
        }
    },
    "attached_files": [
        {
            "filename": "compound_plate_map.pdf",
            "type": "pdf",
            "size": "198 KB"
        },
        {
            "filename": "luminescence_raw.csv",
            "type": "csv",
            "size": "24 KB"
        },
        {
            "filename": "ic50_4PL_prism.xlsx",
            "type": "xlsx",
            "size": "108 KB"
        }
    ],
    "steps": [
        {
            "n": 1,
            "description": "Thaw compound stock + reference inhibitor SCH772984 from -80 °C",
            "duration": "0:10",
            "complete": true,
            "note": "On dry ice, brief vortex"
        },
        {
            "n": 2,
            "description": "Prepare 10-point 3-fold dilution series in DMSO (10 mM → 0.5 µM source)",
            "duration": "0:15",
            "complete": true,
            "note": "Source plate D1"
        },
        {
            "n": 3,
            "description": "Echo-dispense 50 nL per well of 384-well assay plate (4 reps × 10 doses + ctrls)",
            "duration": "0:08",
            "complete": true,
            "note": "Echo 550 acoustic"
        },
        {
            "n": 4,
            "description": "Dispense 5 µL kinase mix (10 nM ERK1 in 1× buffer) per well; pre-incubate 15 min RT",
            "duration": "0:15",
            "complete": true
        },
        {
            "n": 5,
            "description": "Dispense 5 µL substrate + ATP mix (2 µM MBP + 100 µM ATP) per well; start kinase reaction",
            "duration": "0:05",
            "complete": true
        },
        {
            "n": 6,
            "description": "Incubate 60 min at RT (kinase reaction)",
            "duration": "1:00",
            "complete": true
        },
        {
            "n": 7,
            "description": "Dispense 10 µL ADP-Glo Reagent per well; incubate 40 min RT (ADP→ATP)",
            "duration": "0:42",
            "complete": true
        },
        {
            "n": 8,
            "description": "Dispense 20 µL Kinase Detection Reagent per well; incubate 60 min RT (luciferase)",
            "duration": "1:02",
            "complete": true
        },
        {
            "n": 9,
            "description": "Read luminescence on Synergy Neo2 (10 s integration per well); export raw CSV",
            "duration": "0:08",
            "complete": true,
            "note": "BioTek run 2026-0604-1432"
        },
        {
            "n": 10,
            "description": "4PL fit in GraphPad Prism, extract IC50 + Hill + Z' + R²; archive workbook",
            "duration": "0:20",
            "complete": true,
            "note": "IC50 280 nM · slope 1.4 · Z' 0.86 · R² 0.997"
        }
    ],
    "spreadsheet": [
        [
            "Dose (nM)",
            "Rep 1",
            "Rep 2",
            "Rep 3",
            "Rep 4",
            "Mean RLU",
            "% Activity"
        ],
        [
            "10000",
            "12,841",
            "12,503",
            "13,108",
            "12,772",
            "12,806",
            "5"
        ],
        [
            "3333",
            "15,124",
            "14,891",
            "15,308",
            "15,142",
            "15,116",
            "9"
        ],
        [
            "1111",
            "23,008",
            "22,891",
            "23,401",
            "23,099",
            "23,100",
            "18"
        ],
        [
            "370",
            "57,221",
            "58,109",
            "56,892",
            "57,944",
            "57,541",
            "49"
        ],
        [
            "123",
            "90,418",
            "91,287",
            "89,914",
            "90,872",
            "90,623",
            "78"
        ],
        [
            "41",
            "105,891",
            "106,442",
            "104,981",
            "106,182",
            "105,874",
            "90"
        ],
        [
            "14",
            "112,109",
            "111,541",
            "113,008",
            "112,494",
            "112,288",
            "95"
        ],
        [
            "4.5",
            "115,442",
            "114,892",
            "116,201",
            "115,109",
            "115,411",
            "98"
        ],
        [
            "1.5",
            "116,108",
            "115,872",
            "116,991",
            "116,341",
            "116,328",
            "99"
        ],
        [
            "0.5",
            "116,418",
            "115,991",
            "116,712",
            "116,108",
            "116,307",
            "99"
        ],
        [
            "DMSO ctrl",
            "117,108",
            "116,872",
            "117,341",
            "116,994",
            "117,079",
            "100"
        ],
        [
            "no kinase",
            "8,109",
            "7,891",
            "8,341",
            "8,108",
            "8,112",
            "0"
        ]
    ],
    "links": {
        "experiments": [
            {
                "ref_id": "EXP-2026-0529",
                "title": "HTS hit triage — 200 cmpds @ 10 µM",
                "owner": "K. Patel",
                "date": "May 29"
            },
            {
                "ref_id": "EXP-2026-0610",
                "title": "IC50 — analogs 342-347 (SAR series)",
                "owner": "K. Patel",
                "date": "Jun 10"
            }
        ],
        "resources": [
            {
                "ref_id": "SOP-0207",
                "title": "IC50 ADP-Glo Kinase SOP v1.2",
                "owner": "QA Team",
                "date": "Feb 18"
            },
            {
                "ref_id": "KIT-0214",
                "title": "Promega ADP-Glo Kinase Assay insert",
                "owner": "Promega Docs",
                "date": "Mar 12"
            },
            {
                "ref_id": "TGT-ERK1",
                "title": "ERK1 (MAPK3) recombinant kinase datasheet",
                "owner": "Target DB",
                "date": "Jan 8"
            }
        ]
    },
    "compounds": [
        {
            "name": "Recombinant ERK1 (MAPK3), active",
            "catalog_lot": "#PV3311 · Lot 2562842",
            "stock_status": "In stock"
        },
        {
            "name": "MBP substrate (myelin basic protein)",
            "catalog_lot": "#M1891 · Lot SLBS9421A",
            "stock_status": "In stock"
        },
        {
            "name": "ATP (10 mM working stock)",
            "catalog_lot": "in-house · Lot 2026-04",
            "stock_status": "In stock"
        },
        {
            "name": "ADP-Glo Reagent",
            "catalog_lot": "#V6930 · Lot 0000456871",
            "stock_status": "In stock"
        },
        {
            "name": "Kinase Detection Reagent",
            "catalog_lot": "#V6930 · Lot 0000456871",
            "stock_status": "In stock"
        },
        {
            "name": "SCH772984 (reference ERK1/2 inhibitor)",
            "catalog_lot": "#S7101 · Lot SLBT1284",
            "stock_status": "Low"
        }
    ],
    "storage": [
        {
            "name": "Compound source plate (DMSO master)",
            "location": "-80 °C · Compound Mgmt Freezer C-4 · Box CMP-2026-Q2",
            "count": "×1"
        },
        {
            "name": "ERK1 kinase aliquots (10 µL working)",
            "location": "-80 °C · Freezer A-1 · Drawer 2 · Box ENZ-KIN-2026",
            "count": "×24"
        },
        {
            "name": "ADP-Glo reagents (working aliquots)",
            "location": "-20 °C · Freezer A-1 · Drawer 3 · Box ADP-GLO-WK",
            "count": "×6"
        }
    ],
    "permissions": {
        "visibility": [
            {
                "type": "Team",
                "name": "Discovery Biology"
            },
            {
                "type": "Person",
                "name": "K. Patel"
            }
        ],
        "can_write": [
            {
                "type": "Person",
                "name": "K. Patel"
            },
            {
                "type": "Person",
                "name": "Dr. R. Singh"
            }
        ]
    }
}