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TNFRSF25 · Western blot design guide
Design a Western Blot for TNFRSF25
Real validated TNFRSF25 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TNFRSF25 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
TNFRSF25 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 | ~45.4 kDa | |
| Observed band | 59 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | N-linked glycosylation | |
| Regulation | IFN-γ-induced | |
| Isoform | 12 isoform(s) |
Section 1
Real Curated TNFRSF25 Western Blot Protocols
Literature-validated Western blot parameters for TNFRSF25 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | brain samples (mouse) |
| Gel % | 10–12% |
| Transfer | 20% (vol/vol) methanol in NuPage Transfer Buffer at 4°C |
| Membrane | PVDF |
| Primary antibody | 1:1,000 |
| Primary incubation | overnight at 4°C |
| Secondary antibody | 1:4,000 |
| Secondary incubation | 1 h at room temperature |
| Wash | TBS plus 0.1% Tween 20, washed 3 times |
| Detection | enhanced chemiluminescence (ECL) detection system |
Section 2
What Is the Expected TNFRSF25 Western Blot Band Size?
TNFRSF25 has a 45.4 kDa predicted backbone but runs at about 59 kDa on Western blot because N-glycosylation at Asn67/Asn106 outweighs the mass lost to signal peptide cleavage.
| single band near 59 kDa | mature, N-glycosylated full-length TNFRSF25 monomer — the mass added by glycosylation outweighs the small loss from signal peptide cleavage |
| diffuse or smeared band spanning roughly 55-65 kDa | heterogeneous glycan occupancy at Asn67 and Asn106 produces a mixture of glycoforms rather than one discrete mass |
| faint lower band around 42-43 kDa | underglycosylated or immature species close to the signal-peptide-cleaved core protein before full carbohydrate addition |
| extra bands at sizes other than the main ~59 kDa species | distinct alternatively spliced isoforms (12 annotated) contributing differently sized species |
| higher band near roughly double the monomer mass under non-reducing or incompletely denaturing conditions | residual homodimer, formed through death-domain interactions rather than a disulfide bridge, not fully dissociated by the run conditions |
💡Expected TNFRSF25 appearanceExpect a single glycosylated TNFRSF25 band at about 59 kDa in whole-cell lysate, above the 45.4 kDa predicted mass because N-glycosylation at Asn67/Asn106 outweighs the mass lost to signal peptide cleavage.
| Predicted mass (UniProt, 417 aa precursor) | sets the 45.4 kDa baseline; the actual band typically runs higher once glycan mass is added |
| N-glycosylation at Asn67 and Asn106 | adds carbohydrate mass and heterogeneity, lifting the mature band up toward the observed ~59 kDa and potentially broadening it |
| Signal peptide cleavage (residues 1-24) | removes a small N-terminal mass from the precursor before the mature protein is glycosylated, partially offsetting the glycan-driven increase |
| Homodimer formation (death domain-mediated) | can produce a higher-molecular-weight species near double the monomer size if the non-covalent dimer is not fully broken by denaturation, even though it is not disulfide-linked |
| Alternative splice isoforms (12 annotated: 1-12) | differing exon content across isoforms can shift apparent migration or introduce additional bands beyond the main species |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | incomplete reduction or denaturation leaves the non-covalent death-domain-mediated homodimer partly intact | boil samples longer in fresh reducing sample buffer with DTT or beta-mercaptoethanol and confirm resolution on a fully denaturing gel |
| Band lower than expected | a non-glycosylated bacterial recombinant standard or a truncated isoform lacking full extracellular domains is being compared to native protein | compare against a mammalian-expressed lysate control migrating near the 59 kDa native band and confirm isoform identity |
| Broad smear instead of sharp band | heterogeneous N-glycosylation at Asn67 and Asn106 generates a mixture of glycoforms | treat lysate with PNGase F to collapse the smear toward the deglycosylated core mass or use a gradient gel for better resolution |
| Multiple bands | detection of different alternatively spliced isoforms or a mix of monomer and residual homodimer species | verify with an isoform-mapped antibody and confirm which band aligns with the ~59 kDa expected monomer |
| Weak or no signal | low native expression of this single-pass membrane receptor in the sample, or epitope masking by dense glycosylation | enrich the membrane fraction, increase protein loading, or include a deglycosylation step to expose the epitope |
| Fragments below expected size | proteolytic release of ectodomain fragments during lysis or sample handling | add protease inhibitors at lysis and keep samples cold and processed quickly to limit degradation |
Experiment evidence
Western blot images showing TNFRSF25 band
PA2004 · TNFRSF25
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TNFRSF25 Western blot
🧪For positive controls for TNFRSF25 in Western blot, you can use lysate from cells transiently or stably transfected to overexpress TNFRSF25, since no HPA tissue or cell line expression data is available to identify a reliable endogenous positive source.
Positive control: TNFRSF25 overexpression lysate
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin blots alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: As a single-pass type I membrane receptor with no HPA expression data to guide tissue selection, a validated positive control is hard to confirm, so pair an overexpression lysate with siRNA/CRISPR knockdown to establish antibody specificity.
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 TNFRSF25 Western Blot Tips
Deeper troubleshooting and optimisation questions for TNFRSF25, answered from its protein features.
Why does TNFRSF25 run heavier than its predicted 45 kDa?
Predicted mass is 45.4 kDa, but 3 N-glycosylation sites and 12 disulfide bonds in the extracellular domain add mass and alter SDS-PAGE mobility, consistent with the commonly observed ~59 kDa band. This shift is normal for a heavily disulfide-bonded, glycosylated type I membrane receptor and does not indicate a nonspecific band.
Why do TNFRSF25 blots sometimes show multiple bands?
UniProt lists 12 annotated isoforms from alternative splicing, so band pattern and size can vary by tissue or cell line depending on which isoforms are expressed. Compare your lysate source against isoform-specific sequence differences before calling extra bands nonspecific.
Does glycosylation affect TNFRSF25 antibody detection?
TNFRSF25 carries 3 glycosylation sites, which can create migration heterogeneity or mask nearby epitopes on the extracellular domain. If the band appears diffuse or antibody signal is weak, PNGase F deglycosylation prior to loading can sharpen the band and shift it closer to the predicted 45.4 kDa.
What blocking buffer works best for TNFRSF25 blots?
Because TNFRSF25 is a glycoprotein, milk-based blockers (which contain glycoproteins) can increase background via lectin-like cross-reactivity. Use 5% BSA in TBST instead, particularly if the primary antibody targets a glycosylated extracellular epitope.
What transfer method to use for TNFRSF25 Western blot?
Standard wet transfer to PVDF is sufficient given the 45.4 kDa predicted (up to ~59 kDa observed) size. As a single-pass type I transmembrane protein, ensure lysates use a detergent-based buffer for full membrane solubilization before transfer; no extended high-molecular-weight transfer protocol is needed.
Should TNFRSF25 samples be run under reducing conditions?
Yes. TNFRSF25 forms a homodimer and contains 12 disulfide bonds; non-reducing conditions can preserve dimer or disulfide-linked aggregates, producing higher-MW bands. Use DTT or beta-mercaptoethanol and full sample denaturation for consistent monomer-level quantitation at the expected size.
What explains a band near 90-120 kDa on TNFRSF25 blots?
Incomplete reduction can leave the TNFRSF25 homodimer intact, roughly doubling the monomer mass and producing a higher apparent-MW band. Confirm reducing agent concentration and boiling/denaturation time before treating this as nonspecific.
Boster reagents
Best TNFRSF25 Western Blot Antibodies
BosterBio's TNFRSF25 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-DR3/TNFRSF25 Antibody Picoband®
Cat # PA2004
The anti-DR3/TNFRSF25 antibodies below represent Boster's best-performing western blot reagents for this target, extensively cited in the literature and rigorously validated through orthogonal methods, including negative-tissue controls, to ensure specific, reproducible detection.
Which to pick: Only one Boster antibody is listed for TNFRSF25/DR3: PA2004. It includes an actual western blot image (COLO320 whole cell lysate, 0.5ug/ml) showing the expected ~59kD band, so it's the default and only pick—no selection needed.
Source: BosterBio TNFRSF25 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q93038.
- Human Protein Atlas. TNFRSF25 tissue expression.