HOXB9 / Homeobox protein Hox-B9 · Western blot design guide

Design a Western Blot for HOXB9

Source-linked HOXB9 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-HOXB9 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 HOXB9: expected band ~28.1 kDa, hero antibody M05689, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **HOXB9** Western blot protocol sheet — expected band ~28.1 kDa, antibody M05689, controls and PMC citations. Open the full HOXB9 WB guide →

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HOXB9 Western Blot Experimental Design Guide

Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.

Must know before running

	Expected band	~28.1 kDa
	Gel	12–15% (standard starting point)
	Negative control ⓘ	Suggested KO / knockdown lysate

Important caveats

Reasons your observed band may differ from the expected size.

	PTM	Phosphorylated + Ubl conjugation
	Caveat	Modification-state controls
	Gene-set association	MSigDB Hallmark membership
	Isoform	1 isoform(s)

Section 1

Source-Linked HOXB9 Western Blot Protocol Options

The M05689 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	MCF7 cell lysate (catalog M05689)
Gel %	12–15% (standard starting point)
Load	20–30 µg total protein per lane; optimize for abundance (standard starting point)
Transfer	Short semi-dry transfer; verify retention (standard starting point)
Membrane	0.45 µm PVDF (standard starting point)
Blocking	5% milk or 5% BSA in TBST (standard starting point)
Primary antibody	M05689; use the WB datasheet starting dilution (standard starting point)
Primary incubation	Overnight at 4 °C (standard starting point)
Secondary antibody	Species-matched HRP conjugate at validated dilution (standard starting point)
Secondary incubation	1 h at room temperature (standard starting point)
Wash	3 × 5 min in TBST (standard starting point)
Detection	ECL; bracket exposures to avoid saturation (standard starting point)

Section 2

What Is the Expected HOXB9 Western Blot Band Size?

HOXB9 is predicted at 28.1 kDa; phosphorylation and Ubl conjugation could affect migration, but no empirical band size or visible shift is established.

What am I looking at on my blot?
Band near 28.1 kDa	Consistent with the predicted size of HOXB9; confirm its identity with controls
Closely spaced bands near 28.1 kDa	Phosphorylation at Thr133 could contribute, but a visible shift is not established
Band above 28.1 kDa	Ubl conjugation is annotated, but the band’s identity requires confirmation
Weak band in whole-cell lysate	Nuclear HOXB9 may be recovered more effectively in a nuclear fraction

💡Expected HOXB9 appearanceHOXB9 has a predicted mass of 28.1 kDa; no empirical band size is supplied, so identify a candidate band near that size with appropriate controls.

How each factor affects band size
Predicted molecular mass	Places unmodified HOXB9 near 28.1 kDa
Phosphothreonine at Thr133	May affect migration, but no resolvable size change is established
Ubl conjugation annotation	Could produce a higher apparent mass if HOXB9 is conjugated; no band size is established
Nuclear localization	Affects sample recovery rather than intrinsic molecular mass

Why is my band missing or off?
Situation	Likely cause	Next action
No band in lysate	Nuclear HOXB9 may be poorly recovered	Check a nuclear fraction and verify fraction recovery
Band higher than expected	Possible Ubl conjugation or an unidentified band	Confirm HOXB9 identity with a second antibody or HOXB9 depletion
Band lower than expected	A fragment or an unidentified band; no cleavage feature is listed	Check sample integrity and confirm identity by HOXB9 depletion
Multiple bands	Modified HOXB9 species or unrelated antibody binding	Compare bands after HOXB9 depletion; assess phosphorylation if indicated
Weak or no signal	Limited recovery of nuclear HOXB9 or insufficient detectable protein	Check nuclear extraction and include a positive control

Source: UniProt P17482 Human Protein Atlas product datasheet (M05689)

Experiment evidence

Western blot images showing HOXB9 band

M05689 · HOXB9

A05689-1 · HOXB9

← swipe / scroll for more images →

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

Sample controls for HOXB9 Western blot

🧪For positive controls for HOXB9 in Western blot, you can use no HPA-supported tissue or cell sample because none is provided.

Positive control: No high/medium HPA tissue identified

Negative control: Suggested KO / knockdown lysate

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

⚠️Feasibility: HPA data are unavailable, so a positive sample and a clean negative tissue cannot be established.

HPA tissue expression evidence for HOXB9

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
No high/medium HPA tissues identified in the supplied evidence.

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

Tissue	Cell type	Level	Evidence	Source
No lower-expression tissue rows available in the supplied evidence.

Section 3

Advanced HOXB9 Western Blot Tips

Deeper troubleshooting and optimisation questions for HOXB9, answered from its protein features.

How should HOXB9 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.

Are multiple HOXB9 isoforms expected?

Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. Multiple bands should not be assigned to HOXB9 isoforms from this evidence alone.

How should HOXB9 Thr133 phosphorylation be assessed?

PTM · UniProt lists phosphothreonine at position 133 using UniProt sequence numbering. If testing this modification, use a site-specific assay or compare signal after phosphatase treatment. A change in band position alone would not identify Thr133 phosphorylation.

Does this guide establish induction of HOXB9?

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 HOXB9 Western blot?

Transfer · HOXB9 is predicted to be 28.1 kDa. Choose transfer conditions that retain proteins near this size on the membrane, and check transfer with a total-protein stain. The supplied features do not specify an optimal membrane or transfer protocol.

How should blocking be optimized?

Blocking · Standard workflow guidance: follow the M05689 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 nuclear HOXB9 be quantified?

Quantitation · UniProt locates HOXB9 in the nucleus. Quantify bands within the assay’s linear range, use consistent nuclear extraction across samples, and normalize to an appropriate nuclear loading measure.

Does a band above 28.1 kDa indicate modified HOXB9?

Interpretation · Not by itself. UniProt predicts 28.1 kDa and lists phosphothreonine at position 133, but that feature does not establish a visible shift. No observed band size is supplied. Compare the band with a molecular weight marker and verify its identity before assigning a modification.

UniProt includes isopeptide bond and ubiquitin-like conjugation keywords, so modified species are possible. Those keywords do not identify a modification site or establish that an extra band is HOXB9. Verify band identity before attributing it to conjugation.

Boster reagents

HOXB9 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 HOXB9 expression in MCF7 cell lysate.

Anti-HOXB9 Rabbit Monoclonal Antibody

Cat # M05689

View product →

Real WB data

Western blot analysis of HOXB9 using anti-HOXB9 antibody (A05689-1). 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 50ug of sample under reducing conditions. Lane 1: human K562 whole cell lysates, Lane 2: human HEK293 whole cell lysates, Lane 3: human CACO-2 whole cell lysates. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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-HOXB9 antigen affinity purified polyclonal antibody (Catalog # A05689-1) 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 HOXB9 at approximately 32KD. The expected band size for HOXB9 is at 32KD.

Anti-HOXB9 Antibody Picoband®

Cat # A05689-1

View product →

Two the supplier anti-HOXB9 antibodies list human reactivity and show Western blot images: M05689 in MCF7 lysate and A05689-1 in K562, HEK293, and CACO-2 lysates. The supplied evidence is product WB data; no independent validation or publication evidence is provided.

Which to pick: For MCF7 lysate, consider M05689. For K562, HEK293, or CACO-2 lysates, A05689-1 provides documented WB conditions and an approximately 32 kDa band. Choose based on the closest tested sample and protocol.

Source: BosterBio HOXB9 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.

References

UniProt Consortium. UniProt entry P17482.
Human Protein Atlas. HOXB9 tissue expression.
PMC9507699 — target-verified WB comparison
PMC4138122 — target-verified WB comparison
PMC9865712 — target-verified WB comparison