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SFRP2 · Western blot design guide
Design a Western Blot for SFRP2
Real validated SFRP2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SFRP2 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
SFRP2 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 | ~33.5 kDa | |
| Observed band | ~38 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Cleaved | |
| Caveat | Extensive disulfide bonding | |
| Regulation | LPS-induced | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated SFRP2 Western Blot Protocols
Literature-validated Western blot parameters for SFRP2 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human Hela , Lane 2: human A431 , Lane 3: human Caco-2 , Lane 4: human U-87MG . 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-SFRP2 antigen affinity purified polyclonal antibody (Catalog # A01752-1) at 0.25 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 1:10000 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 SFRP2 at approximately 38KD. The expected band size for SFRP2 is at 33KD |
| Gel % | 10–12% |
| Load | 50ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.25 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:10000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 38 kDa |
Section 2
What Is the Expected SFRP2 Western Blot Band Size?
SFRP2 has a 33.5 kDa predicted backbone but runs at ~38 kDa on Western blots, mainly because its eight disulfide bonds create a compact, slow-migrating structure.
| faint or absent band in whole-cell lysate | SFRP2 is a secreted protein, so most of it is exported into conditioned medium rather than retained inside the cell |
| single dominant band around ~38 kDa | this is the empirically observed size for mature secreted SFRP2, running above its 33.5 kDa predicted mass |
| band sitting higher than the 33.5 kDa predicted mass | the eight disulfide bonds lock SFRP2 into a compact, cysteine-dense fold that migrates anomalously slower on SDS-PAGE |
| no smearing or laddering above the main band | SFRP2 has no annotated glycosylation sites, so there is no glycoform heterogeneity to broaden the band |
| single band with no additional isoform-sized species | UniProt lists only one SFRP2 isoform, so no alternate-splicing bands are expected |
| mature band marginally smaller than the full-length translated product | the 24-residue signal peptide is cleaved during secretion, trimming a small amount of mass from the precursor |
💡Expected SFRP2 appearanceExpect a single ~38 kDa band for mature secreted SFRP2, running above its 33.5 kDa predicted mass because of its disulfide-rich, compact fold, with little or no signal in whole-cell lysate.
| Predicted mass (UniProt) | gives a 33.5 kDa baseline for the 295-aa unmodified precursor, below the empirically observed band |
| Signal peptide cleavage (residues 1-24) | removes the N-terminal signal sequence during secretion, making the mature form slightly smaller than the full translated precursor |
| Extensive disulfide bonding (8 bonds) | folds the cysteine-rich domain into a compact structure that migrates anomalously slower, shifting the apparent band above 33.5 kDa toward the observed ~38 kDa |
| Secreted subcellular location | concentrates SFRP2 in conditioned medium/secretome fractions rather than whole-cell lysate, reducing intracellular band intensity |
| Single annotated isoform | predicts one dominant band rather than several isoform-sized species |
| Absence of glycosylation sites | keeps the band sharp and uniform rather than smeared, since there is no glycoform heterogeneity |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | SFRP2 is secreted and largely exported into the extracellular space rather than retained in the cell | probe concentrated conditioned medium or a secreted-protein fraction instead of whole-cell lysate |
| Band higher than expected | the eight disulfide bonds fold SFRP2 into a compact structure that runs anomalously slower than its 33.5 kDa predicted mass | treat the ~38 kDa band as the true reference size and confirm full reduction with fresh reducing agent before assuming the wrong band |
| Band lower than expected | the mature secreted form has already lost its 24-residue signal peptide relative to the full-length translated precursor | confirm whether the antibody or standard was raised against the precursor or the cleaved mature protein |
| Weak or no signal | secreted protein is diluted across a large volume of conditioned medium | concentrate conditioned medium (e.g. by ultrafiltration) and load more total protein per lane |
| Broad smear instead of sharp band | incomplete reduction leaves residual disulfide bonds intact, producing conformational heterogeneity | increase reducing agent concentration and extend heat denaturation before loading |
Experiment evidence
Western blot images showing SFRP2 band
A01752-1 · SFRP2
PB9362 · SFRP2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SFRP2 Western blot
🧪For positive controls for SFRP2 in Western blot, you can use recombinant SFRP2 protein or conditioned medium from cells transfected to overexpress SFRP2, since no Human Protein Atlas tissue expression data are available to identify a naturally high-expressing source.
Positive control: recombinant SFRP2 protein or SFRP2-overexpressing cell conditioned medium
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin antibody controls alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) to confirm equal loading and transfer.
⚠️Feasibility: As a secreted protein with no HPA expression data available, SFRP2 may show little to no signal in standard whole-cell lysates, so conditioned medium or concentrated secretome samples plus a recombinant protein standard are recommended over relying on tissue lysates alone.
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced SFRP2 Western Blot Tips
Deeper troubleshooting and optimisation questions for SFRP2, answered from its protein features.
Why does SFRP2 run higher than its 33.5 kDa predicted mass?
SFRP2 contains 8 disulfide bonds that create a compact, protease-resistant fold; incomplete reduction of these bonds under standard SDS-PAGE conditions can slow migration, producing the ~38 kDa band seen instead of the 33.5 kDa calculated mass. Use fresh DTT or beta-mercaptoethanol and boil samples fully to ensure consistent, complete reduction before loading.
How does signal peptide cleavage affect SFRP2's expected mass?
The N-terminal 24-residue signal peptide is cleaved during secretion, so the mature secreted SFRP2 used for mass calculations is shorter than the full-length precursor. Predicted mass (33.5 kDa) reflects this mature form, yet the protein still runs higher (~38 kDa) on blots, most likely from its disulfide-stabilized tertiary structure rather than residual propeptide, since no propeptide is annotated.
Is SFRP2 expression induced during differentiation or Wnt signaling?
SFRP2 is annotated as a developmental and differentiation-associated protein that modulates the Wnt signaling pathway, so expression often increases during tissue differentiation, remodeling, or when Wnt signaling is activated. Compare lysates or conditioned media from differentiated versus undifferentiated cells, or Wnt-stimulated versus untreated conditions, to assess induction; no specific PTM marks this regulation.
How to optimize blocking for secreted SFRP2 detection?
Because SFRP2 is secreted and often present at low levels, concentrate conditioned media or serum by TCA/acetone precipitation before loading. Use BSA rather than milk for blocking to limit nonspecific background, since SFRP2 lacks glycosylation and antibody cross-reactivity with milk glycoproteins is a common source of noise in secreted-protein blots.
What transfer method to use for SFRP2 Western blot?
Standard wet or semi-dry transfer to PVDF membrane is sufficient for this ~33-38 kDa secreted protein. Run samples under reducing conditions to disrupt the 8 disulfide bonds beforehand, ensuring the protein is fully linearized for efficient, uniform transfer rather than trapped in a disulfide-stabilized globular form that transfers poorly.
How to normalize SFRP2 levels in secreted samples?
Standard housekeeping controls like actin or GAPDH don't apply to conditioned media or serum samples since they are intracellular. Normalize instead to total protein stain (Ponceau or Coomassie), to cell number, or to volume of conditioned media collected under a standardized, serum-free collection protocol to keep comparisons consistent across samples.
What explains extra bands beyond the main SFRP2 band?
With 8 disulfide bonds and no glycosylation, extra higher-molecular-weight bands usually indicate incomplete reduction, leaving disulfide-linked dimers or aggregates rather than glycoform variants. Lower bands likely reflect proteolytic degradation of the secreted protein during collection. Ensure complete reducing conditions and add protease inhibitors during sample collection to minimize these artifacts.
Boster reagents
Best SFRP2 Western Blot Antibodies
BosterBio's SFRP2 antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-SFRP2 Antibody Picoband®
Cat # A01752-1
Real WB data
Anti-SFRP2 Antibody Picoband®
Cat # PB9362
These anti-SFRP2 antibodies are our best-performing, most cited Western blot reagents, thoroughly validated across multiple lysates and orthogonally cross-checked against negative-control tissue and complementary detection methods, giving you confidence in specific, reproducible band detection for your experiments.
Which to pick: Both A01752-1 and PB9362 (Picoband) include real Western blot validation images. PB9362 is shown validated in COLO320 and SW620 whole-cell lysates—pick it if your system matches those lines; otherwise A01752-1's SDS-PAGE-validated result works equally well.
Source: BosterBio SFRP2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q96HF1.
- Human Protein Atlas. SFRP2 tissue expression.