PROX1 / Prospero homeobox protein 1 · Western blot design guide

Design a Western Blot for PROX1

Real validated PROX1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PROX1 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 PROX1: expected band ~83.2 kDa, hero antibody A01985-1, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **PROX1** Western blot protocol sheet — expected band ~83.2 kDa, antibody A01985-1, controls and PMC citations. Open the full PROX1 WB guide →

Navigate to: Protocols Band Controls Antibodies

PROX1 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	~83.2 kDa
	Observed band	~90 kDa
	Gel	10% (catalog A01985-1)
	Negative control ⓘ	Suggested KO / knockdown lysate

Important caveats

Reasons your observed band may differ from the expected size.

	PTM	Phosphorylated + Ubl conjugation
	Caveat	Phosphorylation-state controls
	Gene-set association	MSigDB C7 membership
	Isoform	1 isoform(s)

Section 1

Real Curated PROX1 Western Blot Protocols

The A01985-1 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 HepG2, human HUH7, human SH-SY5Y (catalog A01985-1)
Gel %	10% (catalog A01985-1)
Load	30 ug; reducing conditions (catalog A01985-1)
Transfer	a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A01985-1)
Membrane	nitrocellulose membrane (catalog A01985-1)
Blocking	5% non-fat milk/TBS for 1.5 hour at RT (catalog A01985-1)
Primary antibody	A01985-1 · 0.5 μg/mL (catalog A01985-1)
Primary incubation	overnight at 4°C (catalog A01985-1)
Secondary antibody	goat anti-rabbit IgG-HRP, 1:5000 (catalog A01985-1)
Secondary incubation	1.5 hour at RT (catalog A01985-1)
Wash	TBS-0.1%Tween 3 times with 5 minutes each (catalog A01985-1)
Detection	ECL (catalog A01985-1)

Section 2

What Is the Expected PROX1 Western Blot Band Size?

PROX1 is predicted at 83.2 kDa and observed at ~90 kDa; the cause of the difference is not established.

What am I looking at on my blot?
Band at ~90 kDa	Matches the reported empirical PROX1 band; confirm its identity with antibody controls.
Band near 83.2 kDa	Near the predicted PROX1 mass; band identity still requires confirmation.
Weak band in whole-cell lysate	PROX1 is nuclear; compare with a nuclear-enriched sample.
Additional bands near ~90 kDa	PROX1 has documented phosphoserines, but distinct migrating forms are unproven.

💡Expected PROX1 appearancePROX1 has a predicted mass of 83.2 kDa and a reported empirical band at ~90 kDa; the cause of the difference is unestablished, so confirm band identity with antibody controls.

How each factor affects band size
UniProt predicted mass	Places the sequence-derived reference at 83.2 kDa.
Full-length sequence mass	Corresponds to 83,203 Da before any apparent migration difference.
Predicted mass comparison	The reported ~90 kDa band migrates above the 83.2 kDa prediction; the cause is unestablished.
Predicted mass as an identity reference	A band near 83.2 kDa is plausible but needs independent identity confirmation.

Why is my band missing or off?
Situation	Likely cause	Next action
No band in lysate	Nuclear PROX1 may be underrepresented in the tested preparation.	Compare a nuclear-enriched sample with a positive control lysate.
Band higher than expected	PROX1 is reported at ~90 kDa despite its 83.2 kDa predicted mass; the cause is unknown.	Compare with the reported band and confirm identity using an independent antibody.
Band lower than expected	Band identity or sample integrity is uncertain.	Check a positive control and validate the band with an independent antibody.
Multiple bands	PROX1 has documented phosphoserines, but their effect on band separation is unproven.	Confirm band identity; compare phosphatase-treated samples if investigating phosphorylation.
Weak or no signal	The tested preparation may contain too little nuclear PROX1.	Check loading, nuclear enrichment, and a positive control.

Source: UniProt Q92786 Human Protein Atlas product datasheet (A01985-1)

Experiment evidence

Western blot images showing PROX1 band

A01985-1 · PROX1

PA2229 · PROX1

M01985 · PROX1

M01985-1 · PROX1

← swipe / scroll for more images →

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

Sample controls for PROX1 Western blot

🧪For positive controls for PROX1 in Western blot, you can use an HPA-IHC candidate positive tissue once identified; none is supplied here.

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 such as Ponceau alongside.

⚠️Feasibility: HPA tissue data are absent, so tissue controls cannot be validated from the supplied evidence.

HPA tissue expression evidence for PROX1

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 PROX1 Western Blot Tips

Deeper troubleshooting and optimisation questions for PROX1, answered from its protein features.

How should PROX1 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 PROX1 isoforms explain multiple bands?

Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. It therefore provides no isoform-based explanation for multiple bands. Validate additional bands before assigning them to PROX1.

Which PROX1 modifications matter when interpreting a blot?

PTM · UniProt lists phosphoserines at positions 177, 179, 199, 291, 295, 511, 514, and 557. These are UniProt coordinates; antibody or paper numbering may differ. Their presence does not establish that phosphorylation produces a resolvable band shift.

Does this guide establish induction of PROX1?

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 PROX1 Western blot?

Transfer · PROX1 is predicted at 83.2 kDa, and the supplied observed band is about 90 kDa. Choose and verify a transfer setup that retains proteins in this range, using a molecular weight ladder and a validated control. The supplied features do not specify a particular transfer method.

How should blocking be optimized?

Blocking · Standard workflow guidance: follow the A01985-1 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 nuclear localization affect PROX1 quantitation?

Quantitation · UniProt places PROX1 in the nucleus and notes that RORG promotes its nuclear localization. Compare like sample fractions across conditions, and account for changes in nuclear localization when interpreting differences in band intensity.

Why might PROX1 appear near 90 kDa instead of 83.2 kDa?

Interpretation · PROX1 has a predicted mass of 83.2 kDa, while the supplied observed band is about 90 kDa. UniProt lists phosphorylation, but those features alone do not establish a visible shift or explain the mass difference. Use a molecular weight ladder and a validated PROX1 control to assess band identity.

Check candidate bands against the approximately 90 kDa observed band and validate their identity. The supplied record lists one isoform and no signal peptide or propeptide, so it does not support assigning extra bands to alternative isoforms or peptide cleavage. Listed phosphorylation sites alone cannot identify an unexpected band.

Boster reagents

PROX1 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 PROX1 using anti-PROX1 antibody (A01985-1). Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human HepG2 whole cell lysates, Lane 2: human HUH7 whole cell lysates, Lane 3: human SH-SY5Y whole cell lysates, Lane 4: rat RH35 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-PROX1 antigen affinity purified polyclonal antibody (A01985-1) 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 (Catalog # BA1054) at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for PROX1 at approximately 90 kDa. The expected band size for PROX1 is at 83 kDa.

Anti-PROX1 Antibody Picoband®

Cat # A01985-1

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Real WB data Anti-PROX1 antibody, PA2229, Western blotting Lane 1: Rat Thymus Tissue Lysate Lane 2: HELA Cell Lysate Lane 3: JURKAT Cell Lysate Lane 4: MM231 Cell Lysate

Anti-PROX1 antibody, PA2229, Western blotting Lane 1: Rat Thymus Tissue Lysate Lane 2: HELA Cell Lysate Lane 3: JURKAT Cell Lysate Lane 4: MM231 Cell Lysate

Anti-Prospero homeobox protein 1 PROX1 Antibody Picoband®

Cat # PA2229

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Real WB data Western blot analysis of PROX1 expression in HepG2 cell lysate.

Anti-PROX1 Rabbit Monoclonal Antibody

Cat # M01985

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Real WB data

Western blot analysis of PROX1 using anti-PROX1 antibody (M01985-1). Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human HepG2 whole cell lysates, Lane 2: human SH-SY5Y 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-PROX1 antigen affinity purified monoclonal antibody (M01985-1) 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 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for PROX1 at approximately 90 kDa. The expected band size for PROX1 is at 83 kDa.

Anti-PROX1 Rabbit Monoclonal Antibody

Cat # M01985-1

View product →

Four the supplier anti-PROX1 antibodies have WB images. A01985-1 and M01985-1 report bands near 90 kDa in human lysates, versus an expected 83 kDa; A01985-1 also shows rat RH35. The supplied evidence does not establish independent specificity validation.

Which to pick: For rat samples, A01985-1 shows RH35 lysate and PA2229 shows thymus lysate. For human samples, all four have WB images; match your sample to the pictured cell lines. PA2229 lists mouse reactivity, but its supplied WB caption shows no mouse sample.

Source: BosterBio PROX1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.

References

UniProt Consortium. UniProt entry Q92786.
Human Protein Atlas. PROX1 tissue expression.
PMC5642489 — target-verified WB comparison
PMC8448337 — target-verified WB comparison
PMC3614731 — target-verified WB comparison