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#314 Molecular Biology Running

Western Blot, HER2 vs GAPDH in HeLa vs SK-BR-3

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western-blot HER2 GAPDH breast-cancer protein-validation

Custom ID

This template captures Western blot validation of HER2 (ERBB2) protein expression in breast cancer cell lines.

Goal: confirm differential HER2 expression at the protein level between HeLa (low/no HER2) and SK-BR-3 (high HER2) reference lines, with GAPDH as the loading control.

Method overview: RIPA lysate from each cell line is quantified by BCA, equal protein loaded on a 10% SDS-PAGE gel, transferred to PVDF via semi-dry transfer, blocked in 5% milk, probed overnight with anti-HER2 and anti-GAPDH, detected with HRP-conjugated secondaries and ECL chemiluminescence on the imaging system.

Expected: HER2 band at ~185 kDa, prominent in SK-BR-3 lanes and faint or absent in HeLa lanes. GAPDH band at ~37 kDa equal across all sample lanes.

Last saved: 34 seconds ago

11 fields loaded from Western Blot baseline (WB SOP v2.4)

Sample

Cell line

select

HeLa, SK-BR-3

Lysate prep reference

text

EXP-2026-0904

Lysate concentration

number

2.4mg/mL

Gel & Loading

Gel percentage

number

10%

Loading amount

number

20µg/lane

Ladder

text

PageRuler Plus 250 kDa

Run conditions

text

100V stack, 150V resolve

Transfer & Detection

Transfer method

select

semi-dry, wet

Blocking agent

select

5% milk, 5% BSA

Primary Ab dilution

text

1:1000 o/n 4°C

Secondary Ab dilution

text

1:5000 1h RT

lane_layout.pdf

98 KB

chemidoc_blot_raw.tif

4.8 MB

band_densitometry.xlsx

186 KB

01

Lyse cells in RIPA + protease inhibitors on ice

Recorded at 09:02 by RO

0:25

02

Centrifuge to clear lysate (12k × 10 min, 4°C)

0:12

03

Quantify protein concentration by BCA assay

Plate reader: SpectraMax M5

0:25

04

Prepare samples (Laemmli, denature at 95°C × 5 min)

0:08

05

Load + run 10% SDS-PAGE gel (100V stack, 150V resolve)

Until dye front exits

1:10

06

Semi-dry transfer to PVDF (25V/2.5A, 7 min)

Trans-Blot Turbo

0:10

07

Block in 5% milk/TBST, 1 h at room temperature

1:00

08

Probe with primary antibodies overnight at 4°C

α-HER2 1:1000, α-GAPDH 1:5000

16:00

09

Wash, then incubate with HRP secondaries (1 h, RT)

3 × 10 min TBST wash before + after

1:30

10

ECL detection and imaging on ChemiDoc

Awaiting darkroom

0:08

	A	B	C	D	E	F
1	Lane	Sample	Target	Raw Intensity	Normalized (÷GAPDH)	Fold vs HeLa
2	2	HeLa-1	HER2	1,420	0.18	1.0
3	3	HeLa-2	HER2	1,510	0.19	1.06
4	4	HeLa-3	HER2	1,380	0.17	0.95
5	5	SK-BR-3-1	HER2	37,420	4.62	25.7
6	6	SK-BR-3-2	HER2	38,910	4.81	26.7
7	7	SK-BR-3-3	HER2	36,720	4.55	25.3
8	2-7	all	GAPDH	8,150 avg	1.00 (ref)	na

EXP-2026-0904 Cell lysis, RIPA prep for HeLa & SK-BR-3 R. Okonkwo Mar 11

EXP-2026-0310 qPCR HER2 expression analysis (upstream) Dr. S. Chen Mar 10

SOP-0218 Western Blot Standard Protocol v2.4 QA Team Feb 14

AB-0144 Anti-HER2 mAb (clone TAB250, validated for WB) Antibody DB Dec 8

AB-0011 Anti-GAPDH mAb (loading control set) Antibody DB Aug 22

Anti-HER2 primary (TAB250, mouse mAb)

MA1-13675 · Lot AS2459123

In stock

Anti-GAPDH primary (rabbit mAb)

5174S · Lot 14

In stock

Anti-mouse IgG-HRP secondary

7076S · Lot 38

Low

Anti-rabbit IgG-HRP secondary

7074S · Lot 41

In stock

RIPA lysis buffer + protease inhibitors

89900 · Lot WC318541

In stock

SuperSignal West Pico ECL substrate

34577 · Lot YK382916

In stock

Antibody aliquots (10 µL working)

-20 °C · Antibody Freezer · Rack B · Box AB-22

×24

Cell lysates (50 µL aliquots)

-80 °C · Freezer C-2 · Shelf 1 · Box L18

×12

Visibility

Chen LabR. Okonkwo · Postdoc

Can write

R. Okonkwo · PostdocDr. S. Chen · PI

Color

Stroke width

Eraser

Use Ctrl + click to add text

{
    "experiment": {
        "id": 314,
        "custom_id": "WB-HER2-2026-03",
        "title": "Western Blot, HER2 vs GAPDH in HeLa vs SK-BR-3",
        "template": "Western Blot",
        "template_version": "v3.2",
        "category": "Molecular Biology",
        "status": "Running",
        "started_on": "2026-05-29",
        "tags": [
            "western-blot",
            "HER2",
            "GAPDH",
            "breast-cancer",
            "protein-validation"
        ]
    },
    "main_text": "This template captures Western blot validation of HER2 (ERBB2) protein expression in breast cancer cell lines.\nGoal: confirm differential HER2 expression at the protein level between HeLa (low/no HER2) and SK-BR-3 (high HER2) reference lines, with GAPDH as the loading control.\nMethod overview: RIPA lysate from each cell line is quantified by BCA, equal protein loaded on a 10% SDS-PAGE gel, transferred to PVDF via semi-dry transfer, blocked in 5% milk, probed overnight with anti-HER2 and anti-GAPDH, detected with HRP-conjugated secondaries and ECL chemiluminescence on the imaging system.\nExpected: HER2 band at ~185 kDa, prominent in SK-BR-3 lanes and faint or absent in HeLa lanes. GAPDH band at ~37 kDa equal across all sample lanes.",
    "extra_fields": {
        "Sample": {
            "Cell line": {
                "type": "select",
                "value": "HeLa, SK-BR-3"
            },
            "Lysate prep reference": {
                "type": "text",
                "value": "EXP-2026-0904"
            },
            "Lysate concentration": {
                "type": "number",
                "value": "2.4 mg/mL"
            }
        },
        "Gel & Loading": {
            "Gel percentage": {
                "type": "number",
                "value": "10 %"
            },
            "Loading amount": {
                "type": "number",
                "value": "20 µg/lane"
            },
            "Ladder": {
                "type": "text",
                "value": "PageRuler Plus 250 kDa"
            },
            "Run conditions": {
                "type": "text",
                "value": "100V stack, 150V resolve"
            }
        },
        "Transfer & Detection": {
            "Transfer method": {
                "type": "select",
                "value": "semi-dry, wet"
            },
            "Blocking agent": {
                "type": "select",
                "value": "5% milk, 5% BSA"
            },
            "Primary Ab dilution": {
                "type": "text",
                "value": "1:1000 o/n 4°C"
            },
            "Secondary Ab dilution": {
                "type": "text",
                "value": "1:5000 1h RT"
            }
        }
    },
    "attached_files": [
        {
            "filename": "lane_layout.pdf",
            "type": "pdf",
            "size": "98 KB"
        },
        {
            "filename": "chemidoc_blot_raw.tif",
            "type": "png",
            "size": "4.8 MB"
        },
        {
            "filename": "band_densitometry.xlsx",
            "type": "xlsx",
            "size": "186 KB"
        }
    ],
    "steps": [
        {
            "n": 1,
            "description": "Lyse cells in RIPA + protease inhibitors on ice",
            "duration": "0:25",
            "complete": true,
            "note": "Recorded at 09:02 by RO"
        },
        {
            "n": 2,
            "description": "Centrifuge to clear lysate (12k × 10 min, 4°C)",
            "duration": "0:12",
            "complete": true
        },
        {
            "n": 3,
            "description": "Quantify protein concentration by BCA assay",
            "duration": "0:25",
            "complete": true,
            "note": "Plate reader: SpectraMax M5"
        },
        {
            "n": 4,
            "description": "Prepare samples (Laemmli, denature at 95°C × 5 min)",
            "duration": "0:08",
            "complete": true
        },
        {
            "n": 5,
            "description": "Load + run 10% SDS-PAGE gel (100V stack, 150V resolve)",
            "duration": "1:10",
            "complete": true,
            "note": "Until dye front exits"
        },
        {
            "n": 6,
            "description": "Semi-dry transfer to PVDF (25V/2.5A, 7 min)",
            "duration": "0:10",
            "complete": true,
            "note": "Trans-Blot Turbo"
        },
        {
            "n": 7,
            "description": "Block in 5% milk/TBST, 1 h at room temperature",
            "duration": "1:00",
            "complete": true
        },
        {
            "n": 8,
            "description": "Probe with primary antibodies overnight at 4°C",
            "duration": "16:00",
            "complete": true,
            "note": "α-HER2 1:1000, α-GAPDH 1:5000"
        },
        {
            "n": 9,
            "description": "Wash, then incubate with HRP secondaries (1 h, RT)",
            "duration": "1:30",
            "complete": true,
            "note": "3 × 10 min TBST wash before + after"
        },
        {
            "n": 10,
            "description": "ECL detection and imaging on ChemiDoc",
            "duration": "0:08",
            "complete": false,
            "note": "Awaiting darkroom"
        }
    ],
    "spreadsheet": [
        [
            "Lane",
            "Sample",
            "Target",
            "Raw Intensity",
            "Normalized (÷GAPDH)",
            "Fold vs HeLa"
        ],
        [
            "2",
            "HeLa-1",
            "HER2",
            "1,420",
            "0.18",
            "1.0"
        ],
        [
            "3",
            "HeLa-2",
            "HER2",
            "1,510",
            "0.19",
            "1.06"
        ],
        [
            "4",
            "HeLa-3",
            "HER2",
            "1,380",
            "0.17",
            "0.95"
        ],
        [
            "5",
            "SK-BR-3-1",
            "HER2",
            "37,420",
            "4.62",
            "25.7"
        ],
        [
            "6",
            "SK-BR-3-2",
            "HER2",
            "38,910",
            "4.81",
            "26.7"
        ],
        [
            "7",
            "SK-BR-3-3",
            "HER2",
            "36,720",
            "4.55",
            "25.3"
        ],
        [
            "2-7",
            "all",
            "GAPDH",
            "8,150 avg",
            "1.00 (ref)",
            "na"
        ]
    ],
    "links": {
        "experiments": [
            {
                "ref_id": "EXP-2026-0904",
                "title": "Cell lysis, RIPA prep for HeLa & SK-BR-3",
                "owner": "R. Okonkwo",
                "date": "Mar 11"
            },
            {
                "ref_id": "EXP-2026-0310",
                "title": "qPCR HER2 expression analysis (upstream)",
                "owner": "Dr. S. Chen",
                "date": "Mar 10"
            }
        ],
        "resources": [
            {
                "ref_id": "SOP-0218",
                "title": "Western Blot Standard Protocol v2.4",
                "owner": "QA Team",
                "date": "Feb 14"
            },
            {
                "ref_id": "AB-0144",
                "title": "Anti-HER2 mAb (clone TAB250, validated for WB)",
                "owner": "Antibody DB",
                "date": "Dec 8"
            },
            {
                "ref_id": "AB-0011",
                "title": "Anti-GAPDH mAb (loading control set)",
                "owner": "Antibody DB",
                "date": "Aug 22"
            }
        ]
    },
    "compounds": [
        {
            "name": "Anti-HER2 primary (TAB250, mouse mAb)",
            "catalog_lot": "MA1-13675 · Lot AS2459123",
            "stock_status": "In stock"
        },
        {
            "name": "Anti-GAPDH primary (rabbit mAb)",
            "catalog_lot": "5174S · Lot 14",
            "stock_status": "In stock"
        },
        {
            "name": "Anti-mouse IgG-HRP secondary",
            "catalog_lot": "7076S · Lot 38",
            "stock_status": "Low"
        },
        {
            "name": "Anti-rabbit IgG-HRP secondary",
            "catalog_lot": "7074S · Lot 41",
            "stock_status": "In stock"
        },
        {
            "name": "RIPA lysis buffer + protease inhibitors",
            "catalog_lot": "89900 · Lot WC318541",
            "stock_status": "In stock"
        },
        {
            "name": "SuperSignal West Pico ECL substrate",
            "catalog_lot": "34577 · Lot YK382916",
            "stock_status": "In stock"
        }
    ],
    "storage": [
        {
            "name": "Antibody aliquots (10 µL working)",
            "location": "-20 °C · Antibody Freezer · Rack B · Box AB-22",
            "count": "×24"
        },
        {
            "name": "Cell lysates (50 µL aliquots)",
            "location": "-80 °C · Freezer C-2 · Shelf 1 · Box L18",
            "count": "×12"
        }
    ],
    "permissions": {
        "visibility": [
            {
                "type": "person",
                "name": "Chen Lab"
            },
            {
                "type": "person",
                "name": "R. Okonkwo",
                "title": "Postdoc"
            }
        ],
        "can_write": [
            {
                "type": "person",
                "name": "R. Okonkwo",
                "title": "Postdoc"
            },
            {
                "type": "person",
                "name": "Dr. S. Chen",
                "title": "PI"
            }
        ]
    }
}