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TNFSF10 · Western blot design guide
Design a Western Blot for TNFSF10
Real validated TNFSF10 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TNFSF10 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
TNFSF10 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 | ~32.5 kDa | |
| Observed band | ~35 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Membrane-bound vs soluble form | |
| Regulation | Inflammation up | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated TNFSF10 Western Blot Protocols
Literature-validated Western blot parameters for TNFSF10 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human HL-60 , Lane 2: human THP-1 , 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-TRAIL antigen affinity purified polyclonal antibody (Catalog # A00466-1) 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 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 TRAIL at approximately 35KD. The expected band size for TRAIL 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 | 0.5 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 | 35 kDa |
Section 2
What Is the Expected TNFSF10 Western Blot Band Size?
TNFSF10 has a 32.5 kDa predicted backbone but runs at about 35 kDa on blots, likely due to its type II membrane-anchored structure rather than glycosylation, which it lacks.
| single band at ~35 kDa | the full-length TNFSF10 monomer, running modestly above its 32.5 kDa predicted mass consistent with its type II membrane-anchored structure |
| band near 32.5 kDa | the unmodified TNFSF10 backbone, since the protein carries no annotated glycosylation sites or disulfide-linked oligomerization |
| little or no band in whole-cell lysate | TNFSF10 exists in a soluble, secreted form in addition to the membrane-bound form, so much of the protein pool is released outside the cell rather than retained in lysate |
| two closely spaced bands | expression of the two annotated TNFSF10 splice isoforms at slightly different apparent sizes |
| sharp, non-smeared band | expected given the absence of annotated glycosylation sites, so no glycoform-driven heterogeneity is predicted |
💡Expected TNFSF10 appearanceTNFSF10 typically appears as a band around 35 kDa, modestly above its 32.5 kDa predicted mass, reflecting the non-glycosylated, type II membrane-anchored monomer with no disulfide-linked oligomer.
| Predicted mass from UniProt | sets the 32.5 kDa baseline for the unmodified TNFSF10 monomer backbone |
| Type II membrane signal-anchor sequence | can cause the protein to migrate somewhat higher than predicted, consistent with the ~35 kDa empirically observed band |
| Dual membrane-bound and secreted localization | shifts a portion of the protein pool out of the cell lysate and into the soluble/secreted fraction, reducing lysate band intensity |
| Splice isoforms (isoform 1, isoform 2) | can add a second band of qualitatively different size alongside the principal isoform |
| Absence of annotated glycosylation and disulfide bonds | predicts a single sharp band rather than a glycoform smear or a covalently linked higher-mass complex |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | TNFSF10 exists in a soluble, secreted form that is released from the cell rather than fully retained intracellularly | test concentrated conditioned media or extracellular fractions alongside whole-cell lysate |
| Band higher than expected | the type II membrane-anchored monomer runs above its 32.5 kDa calculated mass | reference the ~35 kDa empirical band reported for this antibody rather than the calculated mass alone |
| Multiple bands | co-expression of the two annotated TNFSF10 splice isoforms in the sample | confirm isoform identity using isoform-specific controls or transcript-level validation |
| Weak or no signal | release of the soluble form into the extracellular space can lower intracellular TNFSF10 abundance | increase lysate loading or enrich membrane fractions, and probe concentrated supernatant separately |
| Broad smear instead of sharp band | unlikely to be glycosylation since none is annotated, so a smear more likely reflects degradation or sample overloading | add protease inhibitors, reduce sample loading, and use fresh lysate |
| Fragments below expected size | the soluble form released from the membrane-bound precursor can appear as a smaller species distinct from the full transmembrane form | expect a smaller band in secreted/conditioned-media fractions separate from the membrane-associated lysate band |
Experiment evidence
Western blot images showing TNFSF10 band
A00466-1 · TNFSF10
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TNFSF10 Western blot
🧪For positive controls for TNFSF10 in Western blot, you can use recombinant TNFSF10 protein, since the Human Protein Atlas provides no tissue or cell line expression data to identify a natural positive source for this protein.
Positive control: Recombinant TNFSF10 protein
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH, β-actin, and a total-protein stain (e.g. stain-free gel, Ponceau S, or REVERT) alongside your target blot.
⚠️Feasibility: TNFSF10 is a type II membrane protein that is also shed as a soluble form, so whole-cell lysates may give weak signal and conditioned medium or a membrane-enriched fraction, plus recombinant protein, should be used to confirm antibody performance since no HPA expression data exists to guide tissue selection.
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 TNFSF10 Western Blot Tips
Deeper troubleshooting and optimisation questions for TNFSF10, answered from its protein features.
Why does TNFSF10 run near 35 kDa instead of 32.5 kDa?
UniProt lists no glycosylation, no disulfides, and no annotated modified residues for TNFSF10, so the ~2.5 kDa shift from the 32.5 kDa predicted mass is most likely a gel-migration effect rather than a PTM. Treat small shifts like this as normal SDS-PAGE variance, not evidence of processing, unless confirmed by mass spec.
Which TNFSF10 isoform is detected on a standard blot?
UniProt annotates two isoforms (1 and 2) via alternative splicing. Most commercial antibodies target sequence shared by isoform 1, the canonical form near 32.5 kDa. If isoform 2 differs in included exons, it may appear as a second, distinct-mass band; confirm which isoform your antibody epitope maps to before interpreting extra bands.
Does phosphorylation affect TNFSF10 apparent molecular weight?
TNFSF10 carries the Phosphoprotein keyword, but the supplied data show zero annotated modified-residue sites, so no specific phosphosite is confirmed. Phosphorylation of this scale would not meaningfully shift a ~32.5 kDa protein on standard SDS-PAGE. Do not expect a resolvable band shift from phosphorylation alone under normal Western blot conditions.
How should blocking be optimized for TNFSF10 detection?
TNFSF10 exists as both a single-pass type II membrane protein and a secreted soluble cytokine, so lysates and conditioned media both need clean, low-background blocking. Use BSA rather than milk when probing near the transmembrane region to avoid nonspecific interactions from milk glycoproteins, and block long enough given the protein's small mass and low glycosylation.
What transfer method to use for TNFSF10 Western blot?
At 32.5 kDa, TNFSF10 transfers efficiently with standard wet or semi-dry transfer; use a 0.2 micron PVDF membrane to retain the small protein and shorter transfer times to prevent blow-through. Because it is membrane-associated and hydrophobic in its transmembrane region, include some SDS in transfer buffer to aid elution from the gel.
Should membrane-bound and soluble TNFSF10 be quantified separately?
UniProt notes TNFSF10 exists both as a cell-membrane-anchored form and a secreted soluble form. Whole-cell lysates capture the membrane form, while conditioned media or serum is needed for the soluble cytokine. Quantify each compartment from the appropriate sample type rather than assuming one blot reflects both pools.
Why might an unexpected band appear near 100 kDa?
TNFSF10 functions as a homotrimer, and its Zinc/Metal-binding annotation indicates zinc coordination stabilizes this trimer. Incomplete denaturation can leave trimeric complexes intact (roughly 3x 32.5 kDa), producing a higher-mass band. Ensure thorough boiling and reducing sample buffer to dissociate trimers into the expected monomeric band.
Boster reagents
Best TNFSF10 Western Blot Antibodies
BosterBio's TNFSF10 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-TRAIL/TNFSF10 Antibody Picoband®
Cat # A00466-1
The anti-TRAIL antibody below is our top-performing pick for TNFSF10 Western blotting, supported by extensive citation history and rigorous validation, including orthogonal confirmation against negative tissue controls and complementary detection methods to ensure specific, reproducible band detection.
Which to pick: Only one Boster antibody is catalogued for TNFSF10: A00466-1, which includes a validated Western blot image showing a specific TRAIL band on SDS-PAGE, making it the clear, ready-to-use choice for this target.
Source: BosterBio TNFSF10 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P50591.
- Human Protein Atlas. TNFSF10 tissue expression.