NOTCH1 · Western blot design guide

NOTCH1 Western Blot Planning Guide

Plan a NOTCH1 Western blot around the catalog-observed 272.5 kDa band, image-backed A00033-2 evidence, HPA controls, and verified protocol records.

Evidence assembled July 2026 · For research use; verify linked source records and product datasheet before use

Western blot protocol sheet for NOTCH1 (NOTCH1): expected band 272.5 kDa, antibody A00033-2, and PMC-cited SDS-PAGE protocol steps — NOTCH1 Western blot protocol sheet — expected band 272.5 kDa, antibody A00033-2, controls and PMC citations. Open the full NOTCH1 WB guide →

Navigate to: Protocols Band Controls Antibodies

NOTCH1 Western Blot Experimental Design Guide

Expected bands, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.

Must know before running

	Expected band	272.5 kDa
	Observed band	Not reported — verify product WB image
	Gel	4-12% gradient
	Positive control ⓘ	Gallbladder
	Negative control ⓘ	Ovary

Important caveats

Reasons your observed band may differ from the expected size.

ⓘ	Calculated mass	272.5 kDa
ⓘ	Localization	Cell membrane / Late endosome membrane
ⓘ	Processing / PTM	Record-dependent
ⓘ	Reactivity	Human / Mouse

Section 1

Real Curated NOTCH1 Western Blot Protocols

Start with the molecular-weight rule, then compare verified publication-derived conditions.

Recommended Western blot protocol parameters
Sample / lysate	Adrenal gland
Gel %	4-12% gradient
Load	20-30 µg total protein per lane
Transfer	Wet/tank, extended transfer
Membrane	0.45 µm PVDF
Blocking	5% non-fat milk or 5% BSA in TBST
Primary	A00033-2 at datasheet starting dilution
Primary incubation	Overnight at 4 °C with gentle agitation
Secondary	Species-matched HRP conjugate at validated dilution
Wash	3 × 5 min in TBST
Detection	Chemiluminescent substrate
Exposure	Bracket exposures to avoid saturation

Section 2

What Is the Expected NOTCH1 Western Blot Band Size?

Use the product-observed 272.5 kDa band as the primary planning value and retain the UniProt calculated mass as context.

What am I looking at on my blot?
272.5 kDa	Matches the authoritative product WB observation.
272.5 kDa calculated	Use as UniProt context, not as a replacement observed band.
Unexpected additional signal	Do not assign identity without orthogonal positive/negative controls.

💡Expected NOTCH1 appearancePlan around 272.5 kDa and keep the calculated mass as supporting context.

How each factor affects band size
Catalog-observed band	272.5 kDa; use this as the primary experimental expectation.
Calculated mass	272.5 kDa from UniProt P46531; retain as context.
Gel selection	4-12% gradient; shared with the recommended protocol and poster.
Specificity check	Compare the lead HPA positive and negative controls with A00033-2.

Why is my band missing or off?
Situation	Likely cause	Next action
272.5 kDa	Matches the authoritative product WB observation.	Confirm with orthogonal controls and the linked product record.
Additional band	May reflect processing, modification, or non-specific signal.	Run a dilution series and compare positive/negative controls.
Weak signal	Target abundance or transfer may be limiting.	Verify transfer, increase positive-control abundance, and bracket exposure.

Sources: UniProt P46531 Human Protein Atlas product datasheet (A00033-2)

Experiment evidence

Western blot images showing NOTCH1 band

A00033-2 · NOTCH1

← swipe / scroll for more images →

Real WB data behind the band-size expectations in this section.

Sample controls for NOTCH1 Western blot

🧪Use Gallbladder as the first positive-control candidate and Ovary as the HPA Not detected negative candidate.

Positive control: Gallbladder (High)

Negative control: Ovary (Not detected)

HPA protein score determines control status; other expression data is supporting context only.

HPA tissue expression evidence for NOTCH1

Comprehensive Human Protein Atlas IHC scoring per tissue. Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.

Positive expression · recommended positive controls

Tissue	Cell type	Level	Evidence	Source
Gallbladder	Reported tissue cells	High	Protein (HPA)	HPA →
Cerebellum	Reported tissue cells	High	Protein (HPA)	HPA →
Adrenal gland	Reported tissue cells	High	Protein (HPA)	HPA →
Appendix	Reported tissue cells	High	Protein (HPA)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Ovary	Reported tissue cells	Not detected	Protein (HPA)	HPA →

Section 3

Advanced NOTCH1 Western Blot Tips

Deeper troubleshooting and optimisation questions for NOTCH1, answered from its protein features.

Which band should guide the blot?

Use 272.5 kDa, the observation attached to the authoritative A00033-2 WB record.

How should calculated mass be interpreted?

Treat the UniProt calculated mass as context; it does not replace the catalog-observed 272.5 kDa expectation.

Which positive control should I start with?

Start with Gallbladder, the lead HPA protein-expression candidate.

Which negative control is defensible?

Use Ovary as an orthogonal HPA Not detected candidate.

Which gel should I use?

Use 4-12% gradient consistently across the quick facts, protocol table, and poster.

What transfer method to use for NOTCH1 Western blot?

Use the transfer method in the recommended protocol and verify transfer before blocking.

How should A00033-2 be started?

Start at the linked datasheet condition and run a three-point primary-antibody dilution test.

Which publication-derived protocols can I compare?

Compare only the supplied target-verified records: PMC6675521, PMC4619967.

Boster reagents

NOTCH1 Western Blot Reagents

Human/Mouse-reactive NOTCH1 Western blot reagents with authoritative product imagery.

Real WB data Western blot validation image for NOTCH1 using A00033-2; observed band 272.5 kDa

Anti-Notch1 Antibody Picoband®

Cat # A00033-2

View product →

Real WB data Western blot validation image for NOTCH1 using PA1215; observed band 272.5 kDa