PFN3 / Profilin-3 · Western blot design guide

Design a Western Blot for PFN3

Source-linked PFN3 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PFN3 WB antibodies. Everything you need to plan the experiment before you commit precious samples.

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

Western blot protocol sheet for PFN3: expected band ~14.6 kDa, hero antibody A13729, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **PFN3** Western blot protocol sheet — expected band ~14.6 kDa, antibody A13729, controls and PMC citations. Open the full PFN3 WB guide →

Navigate to: Protocols Band Controls Antibodies

PFN3 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	~14.6 kDa
	Observed band	15 kDa
	Gel	5–20% (catalog A13729)
	Negative control ⓘ	Suggested KO / knockdown lysate

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

Source-Linked PFN3 Western Blot Protocol Options

The A13729 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	rat C6, mouse RAW264.7 (catalog A13729)
Gel %	5–20% (catalog A13729)
Load	30 ug; reducing conditions (catalog A13729)
Transfer	a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A13729)
Membrane	nitrocellulose membrane (catalog A13729)
Blocking	5% non-fat milk/TBS for 1.5 hour at RT (catalog A13729)
Primary antibody	A13729 · 0.5 μg/mL (catalog A13729)
Primary incubation	overnight at 4°C (catalog A13729)
Secondary antibody	goat anti-rabbit IgG-HRP, 1:5000 (catalog A13729)
Secondary incubation	1.5 hour at RT (catalog A13729)
Wash	TBS-0.1%Tween 3 times with 5 minutes each (catalog A13729)
Detection	ECL (catalog A13729)

Section 2

What Is the Expected PFN3 Western Blot Band Size?

PFN3 is predicted at 14.6 kDa and observed at approximately 15 kDa; the cause of the small difference is not established.

What am I looking at on my blot?
Single band near 15 kDa	Consistent with the observed PFN3 band; confirm identity with controls
Single band near 14.6 kDa	Consistent with the predicted mass of PFN3
Additional bands above 15 kDa	Their identity as PFN3 species is unestablished
Bands below 15 kDa	Their identity as PFN3 fragments is unestablished

💡Expected PFN3 appearancePFN3 has a predicted mass of 14.6 kDa and an observed band at approximately 15 kDa; confirm band identity with appropriate controls.

How each factor affects band size
UniProt predicted mass	Sets the full-length reference at 14.6 kDa
14596 Da sequence mass	Corresponds to approximately 14.6 kDa
137-residue full-length chain	Defines the sequence used for the predicted mass
Single annotated protein form	Provides no annotated alternative sequence mass

Why is my band missing or off?
Situation	Likely cause	Next action
No band in lysate	PFN3 signal may be below detection	Check lysate loading and include a positive control
Band higher than expected	Identity of the higher band is unestablished	Compare with the 15 kDa control band and verify specificity
Band lower than expected	Identity of the lower band is unestablished	Check sample integrity and verify specificity
Multiple bands	Additional bands have no annotated PFN3 isoform explanation	Use a positive control and assess antibody specificity
Weak or no signal	PFN3 abundance may be below detection	Check loading, transfer, and the positive control

Source: UniProt P60673 Human Protein Atlas product datasheet (A13729)

Experiment evidence

Western blot images showing PFN3 band

A13729 · PFN3

← swipe / scroll for more images →

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

Sample controls for PFN3 Western blot

🧪For positive controls for PFN3 in Western blot, you can use no HPA-supported tissue or cell sample from the supplied evidence.

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 has no expression data here, so suitable tissue controls cannot be identified.

HPA tissue expression evidence for PFN3

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 PFN3 Western Blot Tips

Deeper troubleshooting and optimisation questions for PFN3, answered from its protein features.

How should PFN3 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 PFN3 isoforms explain multiple bands?

Isoforms · UniProt lists one PFN3 isoform and no alternative sequence. Its supplied features therefore do not support assigning multiple bands to PFN3 isoforms.

Are PFN3 modifications expected to shift its band?

PTM · The supplied features list no modified residues or glycosylation sites. Do not assign a band shift to a specific PFN3 modification from this record.

Does this guide establish induction of PFN3?

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 PFN3 Western blot?

Transfer · PFN3 is a small protein at 14.6 kDa. Choose transfer conditions validated for retaining proteins near 15 kDa, and check transfer and retention with a marker in that range.

How should blocking be optimized?

Blocking · Standard workflow guidance: follow the A13729 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 PFN3 localization affect quantitation?

Quantitation · UniProt places PFN3 in the cytoplasm, cytoskeleton, and nucleus. Compare samples prepared with the same extraction or fractionation method; different recovery from these compartments can affect the measured signal.

Does a 15 kDa band match PFN3’s predicted mass?

Interpretation · Yes. The observed 15 kDa band is close to PFN3’s predicted 14.6 kDa mass. This small difference does not establish a modification or processing event.

UniProt reports an interaction with ACTRT3, but that interaction alone does not identify a higher Western-blot band. The record lists no propeptide, signal peptide, or alternative sequence that explains additional PFN3 bands; verify their identity before assigning them to PFN3.

Boster reagents

PFN3 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 PFN3 using anti-PFN3 antibody (A13729). 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: rat C6 whole cell lysates, Lane 2: mouse 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-PFN3 antigen affinity purified polyclonal antibody (Catalog # A13729) 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 PFN3 at approximately 15 kDa. The expected band size for PFN3 is at 15 kDa.

Anti-PFN3 Antibody Picoband®

Cat # A13729

View product →

A13729 is a rabbit polyclonal anti-PFN3 antibody listed for human, mouse, and rat. Its Western blot image shows an approximately 15 kDa band in rat C6 and mouse RAW264.7 whole cell lysates. The supplied evidence does not show a human sample or independent validation.

Which to pick: A13729 is the only listed option. Its Western blot image uses rat C6 and mouse RAW264.7 whole cell lysates at 0.5 μg/mL; human reactivity is listed, but no human Western blot example is supplied.

Source: BosterBio PFN3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.

References

UniProt Consortium. UniProt entry P60673.
Human Protein Atlas. PFN3 tissue expression.
PMC2694148 — target-verified WB comparison