HES5 / Transcription factor HES-5 · Western blot design guide

Design a Western Blot for HES5

Real validated HES5 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-HES5 WB antibodies. Everything you need to plan the experiment before you commit precious samples.

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

Western blot protocol sheet for HES5: expected band ~18.2 kDa, hero antibody A05384-1, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **HES5** Western blot protocol sheet — expected band ~18.2 kDa, antibody A05384-1, controls and PMC citations. Open the full HES5 WB guide →

Navigate to: Protocols Band Controls Antibodies

HES5 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	~18.2 kDa
	Observed band	~18 kDa
	Gel	5–20% (catalog A05384-1)
	Positive control ⓘ	Caudate (IHC candidate; verify WB) +4 more
	Negative control ⓘ	Adipose tissue (IHC candidate; verify WB)

Important caveats

Reasons your observed band may differ from the expected size.

	PTM	—
	Caveat	—
	Gene-set association	MSigDB C7 membership
	Isoform	1 isoform(s)

Section 1

Real Curated HES5 Western Blot Protocols

The A05384-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.

Recommended Western blot protocol parameters
Sample / lysate	human Hacat, human HepG2 (catalog A05384-1)
Gel %	5–20% (catalog A05384-1)
Load	30 ug; reducing conditions (catalog A05384-1)
Transfer	a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A05384-1)
Membrane	nitrocellulose membrane (catalog A05384-1)
Blocking	5% non-fat milk/TBS for 1.5 hour at RT (catalog A05384-1)
Primary antibody	A05384-1 · 0.5 μg/mL (catalog A05384-1)
Primary incubation	overnight at 4°C (catalog A05384-1)
Secondary antibody	goat anti-rabbit IgG-HRP, 1:5000 (catalog A05384-1)
Secondary incubation	1.5 hour at RT (catalog A05384-1)
Wash	TBS-0.1%Tween 3 times with 5 minutes each (catalog A05384-1)
Detection	ECL (catalog A05384-1)

Section 2

What Is the Expected HES5 Western Blot Band Size?

HES5 is predicted at 18.2 kDa and observed at ~18 kDa; the supplied features establish no cause for their small difference.

What am I looking at on my blot?
Band at ~18 kDa in whole-cell lysate	Matches the reported HES5 band and its 18.2 kDa predicted mass
~18 kDa band enriched in a nuclear fraction	Consistent with HES5 nuclear localization
Weak or absent band in a cytoplasmic fraction	Consistent with HES5 nuclear localization
Single sharp band near 18 kDa	Consistent with the predicted mass and no listed size-changing modifications

💡Expected HES5 appearanceHES5 has a predicted mass of 18.2 kDa and a reported band at ~18 kDa in reducing whole-cell blots; confirm band identity with appropriate controls.

How each factor affects band size
UniProt predicted mass	Places full-length HES5 near 18.2 kDa
Calculated mass of 18,226 Da	Corresponds to approximately 18.2 kDa
Full-length sequence mass	Provides the reference for assessing apparent band size
Predicted 18.2 kDa band position	Agrees closely with the reported ~18 kDa band

Why is my band missing or off?
Situation	Likely cause	Next action
No band in lysate	A cytoplasmic preparation may lack nuclear HES5	Check a nuclear fraction and an extraction control
Band higher than expected	The supplied features do not establish a size-increasing modification	Compare with the ~18 kDa reference and verify identity with an independent antibody
Band lower than expected	The supplied features do not establish a cleaved HES5 product	Check sample integrity and verify identity with an independent antibody
Multiple bands	No named alternative isoforms or size-changing modifications are listed	Check specificity against an HES5-depleted sample
Weak or no signal	HES5 is nuclear and may be underrepresented in the tested fraction	Check nuclear extraction and include a positive-control sample

Source: UniProt Q5TA89 Human Protein Atlas product datasheet (A05384-1)

Experiment evidence

Western blot images showing HES5 band

A05384-1 · HES5

A05384 · HES5

M05384 · HES5

← swipe / scroll for more images →

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

Sample controls for HES5 Western blot

🧪HPA-IHC candidate guidance (verify in WB): For positive controls for HES5 in Western blot, you can use caudate tissue, where HPA reports high expression.

Positive control: Caudate (IHC candidate; verify WB)

Negative control: Adipose tissue (IHC candidate; verify WB)

Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.

⚠️Feasibility: Because HES5 is nuclear, a nuclear extract may improve detection.

HPA tissue expression evidence for HES5

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.

Higher expression tissues · candidate positive controls from IHC

Tissue	Cell type	Level	Evidence	Source
Caudate	neuronal cells	High	Protein (IHC)	HPA →
Cerebral cortex	neuronal cells	High	Protein (IHC)	HPA →
Hippocampus	neuronal cells	High	Protein (IHC)	HPA →
Bronchus	respiratory epithelial cells	Medium	Protein (IHC)	HPA →
Cerebellum	cells in molecular layer	Medium	Protein (IHC)	HPA →

Lower expression tissues · IHC evidence, not confirmed WB-negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	adipocytes	Not detected	Protein (IHC)	HPA →
Appendix	glandular cells	Not detected	Protein (IHC)	HPA →
Bone marrow	hematopoietic cells	Not detected	Protein (IHC)	HPA →
Breast	adipocytes	Not detected	Protein (IHC)	HPA →
Cervix	squamous epithelial cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced HES5 Western Blot Tips

Deeper troubleshooting and optimisation questions for HES5, answered from its protein features.

How should HES5 band migration be interpreted?

Band shift · Use the separately labelled calculated mass and catalog-observed evidence above. A sequence annotation does not establish an observed migration shift. Verify target identity with orthogonal controls.

Could annotated HES5 isoforms explain multiple bands?

Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. It provides no isoform-based explanation for multiple bands; confirm unexpected bands with an independent HES5-specific check.

Do annotated modifications prove a band shift?

PTM · The linked UniProt record describes protein features. A modification annotation alone does not demonstrate a visible shift; retain any condition or experimental qualifier attached to it.

Does this guide establish induction of HES5?

Induction · No general induction response is established by this guide. A pathway or gene-set association is not evidence of induction in a particular specimen. Verify the relevant treatment and control in a target-specific experiment.

What transfer method to use for HES5 Western blot?

Transfer · HES5 is a small, 18.2-kDa protein. Use a transfer setup suited to retaining small proteins, such as a 0.2-µm membrane, and check both transfer and membrane retention.

How should blocking be optimized?

Blocking · Standard workflow guidance: follow the A05384-1 datasheet where specified. Otherwise compare 5% milk or 5% BSA in TBST; for a phospho-specific assay start with BSA. Optimize background and specific signal with matched controls.

How should HES5 bands be quantified?

Quantitation · Quantify the band near the supplied observed position of 18 kDa. If using nuclear extracts, normalize against a suitable nuclear loading reference and compare samples prepared the same way.

Should HES5 migrate near its predicted mass?

Interpretation · HES5 is predicted at 18.2 kDa, and the supplied observed band is approximately 18 kDa. This agreement supports the expected band assignment, but mass alone does not establish identity.

UniProt places HES5 in the nucleus. Include the nuclear fraction when preparing samples, and check fraction recovery before interpreting a weak or absent band.

The record lists no modified residues, glycosylation sites, or alternative sequence. It notes a functional complex with a Groucho/TLE corepressor, but does not show that this complex survives Western blot preparation. These features alone cannot assign an unexpected band or demonstrate a visible mass shift.

Boster reagents

HES5 Western Blot Antibodies

Catalog antibodies with Western blot application and product-specific WB images. Evaluate suitability with the reported sample, controls and experimental conditions.

Real WB data

Western blot analysis of HES5 using anti-HES5 antibody (A05384-1). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human Hacat whole cell lysates, Lane 2: human HepG2 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-HES5 antigen affinity purified polyclonal antibody (Catalog # A05384-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for HES5 at approximately 18 kDa. The expected band size for HES5 is at 18 kDa.

Anti-HES5 Antibody Picoband®

Cat # A05384-1

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Real WB data Western blot analysis of HES5 in Raji cell lysate with HES5 antibody at 1 μg/ml (A) the absence and (B) the presence of blocking peptide.

Anti-Transcription factor HES-5 HES5 Antibody

Cat # A05384

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Real WB data

Western blot analysis of HES5 using anti-HES5 antibody (M05384). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human U251 whole cell lysates, Lane 2: rat spleen tissue lysates, Lane 3: human RAW264.7 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-HES5 antigen affinity purified monoclonal antibody (M05384) at 1:500 overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:500 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for HES5 at approximately 18 kDa. The expected band size for HES5 is at 18 kDa.

Anti-HES5 Monoclonal Antibody

Cat # M05384

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Three the supplier anti-HES5 antibodies have Western blot images. A05384-1 shows an approximately 18 kDa band in human HaCaT and HepG2 lysates; M05384 shows a similar band in reported lysates. A05384 shows a Raji lysate blot with a blocking peptide comparison. These examples do not establish universal species validation.

Which to pick: For human HaCaT or HepG2 samples, consider A05384-1. For a Raji lysate example with a blocking peptide comparison, consider A05384. M05384 has an image including U251 and rat spleen samples. A05384 and M05384 list human, mouse and rat reactivity; A05384-1 lists human only.

Source: BosterBio HES5 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.

References

UniProt Consortium. UniProt entry Q5TA89.
Human Protein Atlas. HES5 tissue expression.
PMC5879899 — target-verified WB comparison
PMC5385399 — target-verified WB comparison
PMC4057709 — target-verified WB comparison