SPOP / Speckle-type POZ protein · Western blot design guide

Design a Western Blot for SPOP

Real validated SPOP Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SPOP 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 SPOP: expected band ~42.1 kDa, hero antibody M02032, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **SPOP** Western blot protocol sheet — expected band ~42.1 kDa, antibody M02032, controls and PMC citations. Open the full SPOP WB guide →

Navigate to: Protocols Band Controls Antibodies

SPOP 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	~42.1 kDa
	Observed band	~42 kDa
	Gel	10% (catalog M02032)
	Negative control ⓘ	Suggested KO / knockdown lysate

Important caveats

Reasons your observed band may differ from the expected size.

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

Section 1

Real Curated SPOP Western Blot Protocols

The M02032 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 Caco-2, human K562 (catalog M02032)
Gel %	10% (catalog M02032)
Load	30 ug; reducing conditions (catalog M02032)
Transfer	a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M02032)
Membrane	nitrocellulose membrane (catalog M02032)
Blocking	5% non-fat milk/TBS for 1.5 hour at RT (catalog M02032)
Primary antibody	M02032 · 1:500 (catalog M02032)
Primary incubation	overnight at 4°C (catalog M02032)
Secondary antibody	goat anti-rabbit IgG-HRP, 1:5000 (catalog M02032)
Secondary incubation	1.5 hour at RT (catalog M02032)
Wash	TBS-0.1%Tween 3 times with 5 minutes each (catalog M02032)
Detection	ECL (catalog M02032)

Section 2

What Is the Expected SPOP Western Blot Band Size?

SPOP is predicted at 42.1 kDa and observed at ~42 kDa; the cause of the small difference is not established.

What am I looking at on my blot?
Band at ~42 kDa	Matches the observed SPOP band and its 42.1 kDa predicted monomer mass.
Band near ~84 kDa	Could reflect a SPOP homodimer that survives sample preparation; this migration is not demonstrated.
Several higher bands	Could reflect retained SPOP homooligomers; confirm their identity.
Weak band in a cytoplasmic fraction	May reflect SPOP localization to the nucleus and nuclear speckles.

💡Expected SPOP appearanceSPOP is predicted at 42.1 kDa and observed at ~42 kDa in reducing whole-cell lysates; confirm band identity with an independent antibody or SPOP depletion.

How each factor affects band size
Predicted monomer mass	Places full-length SPOP near 42.1 kDa, consistent with the observed ~42 kDa band.
SPOP homodimer	Could produce a band near twice the monomer mass if it survives denaturation.
SPOP homooligomer	Could produce higher bands if oligomers remain intact during electrophoresis.
SPOPL heterodimer	Could produce a higher complex band if retained; its apparent size is not supplied.

Why is my band missing or off?
Situation	Likely cause	Next action
No band in lysate	Nuclear SPOP may be poorly recovered.	Check nuclear extraction and use a validated positive lysate.
Band higher than expected	A SPOP dimer or oligomer may have survived denaturation.	Compare adequately denatured samples and verify identity with SPOP depletion.
Band lower than expected	The supplied features do not establish a smaller SPOP form.	Check sample integrity and test whether the band disappears after SPOP depletion.
Multiple bands	Retained oligomers or unrelated antibody binding may contribute.	Compare denaturation conditions and verify bands with an independent antibody.
Weak or no signal	Nuclear SPOP may be underrepresented in the prepared sample.	Check extraction and loading with a nuclear marker.
Fragments below expected size	The supplied features do not establish a specific SPOP cleavage product.	Limit sample degradation and verify fragments with an independent antibody.

Source: UniProt O43791 Human Protein Atlas product datasheet (M02032)

Experiment evidence

Western blot images showing SPOP band

M02032 · SPOP

A02032-2 · SPOP

← swipe / scroll for more images →

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

Sample controls for SPOP Western blot

🧪For positive controls for SPOP in Western blot, you can use a validated SPOP-positive lysate; the supplied HPA data identify no tissue or cell line.

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 the samples.

⚠️Feasibility: With no HPA expression data, tissue controls cannot be selected, and ubiquitous expression is unconfirmed.

HPA tissue expression evidence for SPOP

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 SPOP Western Blot Tips

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

How should SPOP 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.

Should SPOP isoforms produce separate bands?

Isoforms · Only one isoform is listed, with no alternative sequence. These features provide no basis for assigning separate bands to SPOP isoforms.

Could an annotated modification explain a shifted SPOP band?

PTM · No modified residues or glycosylation sites are listed. The Ubl conjugation pathway keyword does not identify a modification site on SPOP. A shifted band cannot be assigned to a specific modification from these features.

Does this guide establish induction of SPOP?

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.

How should transfer be checked for SPOP?

Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.

How should blocking be optimized?

Blocking · Standard workflow guidance: follow the M02032 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 SPOP be quantified?

Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.

Does the observed SPOP band match its predicted mass?

Interpretation · The observed band is approximately 42 kDa, close to the predicted 42.1 kDa for the 374-residue protein. The supplied features do not establish a cause for any small mobility difference.

SPOP is reported to form homodimers and homooligomers and to heterodimerize with SPOPL. These interactions make an associated species worth considering, but the features alone do not show that it survives sample preparation or produces a visible band.

SPOP is reported in the nucleus, including nuclear speckles, and in the cytoplasm. Consider both fractions when interpreting a weak or absent band in either fraction alone.

Compare the approximately 42 kDa band in equivalent sample fractions. Because SPOP is reported in both nucleus and cytoplasm, a change in one fraction alone need not represent the same change in total cellular SPOP.

Start with the approximately 42 kDa band expected from the predicted mass and observed result. The single listed isoform and absence of annotated modified residues do not support assigning other bands to isoforms or specific modifications. Report those bands as unassigned unless independently verified.

Boster reagents

SPOP 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 SPOP using anti-SPOP antibody (M02032). 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 Caco-2 whole cell lysates, Lane 2: human K562 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-SPOP antigen affinity purified monoclonal antibody (M02032) 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 SPOP at approximately 42 kDa. The expected band size for SPOP is at 42 kDa.

Anti-SPOP Rabbit Monoclonal Antibody

Cat # M02032

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Real WB data

Western blot analysis of SPOP using anti-SPOP antibody (A02032-2). 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 unde r reducing conditions. Lane 1: human PC-3 whole cell lysates, Lane 2: human Hela whole cell lysates, Lane 3: human HepG2 whole cell lysates, Lane 4: human MOLT-4 whole cell lysates, Lane 5: human K562 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-SPOP antigen affinity purified polyclonal antibody (Catalog # A02032-2) 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 SPOP at approximately 50 kDa. The expected band size for SPOP is at 42 kDa.

Anti-SPOP Antibody Picoband®

Cat # A02032-2

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Both listed anti-SPOP antibodies have human Western blot images. M02032 shows an approximately 42 kDa band in Caco-2 and K562 lysates. A02032-2 shows an approximately 50 kDa band across five human cell lines, above the stated 42 kDa expectation.

Which to pick: For a band near the expected 42 kDa, start with M02032. Consider A02032-2 if its tested cell lines better match your sample, while accounting for its reported 50 kDa band. Both list human reactivity only.

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

References

UniProt Consortium. UniProt entry O43791.
Human Protein Atlas. SPOP tissue expression.
PMC10333085 — target-verified WB comparison
PMC5887163 — target-verified WB comparison
PMC4172815 — target-verified WB comparison