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TNFSF14 / Tumor necrosis factor ligand superfamily member 14 · Western blot design guide
Design a Western Blot for TNFSF14
Real validated TNFSF14 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TNFSF14 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
TNFSF14 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 | ~26.4 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated | |
| Caveat | Glycosylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated TNFSF14 Western Blot Protocols
The A03516 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Target-positive lysate and matched negative control (standard starting point) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Short semi-dry transfer; verify retention (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A03516; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Species-matched HRP conjugate at validated dilution (standard starting point) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL; bracket exposures to avoid saturation (standard starting point) |
Section 2
What Is the Expected TNFSF14 Western Blot Band Size?
TNFSF14 has a predicted mass of 26.4 kDa; glycosylation, isoforms, and homotrimer formation are annotated, but their effects on blot migration are unproven.
| Band near 26.4 kDa | Consistent with the predicted mass of TNFSF14; confirm band identity with controls |
| Band above 26.4 kDa | N-linked glycosylation at Asn102 could affect migration; the size of any shift is unknown |
| High-mass band | Could reflect retained TNFSF14 homotrimers; their migration on the blot is not established |
| Multiple bands | Isoforms 1 and 2 are annotated, but distinct resolvable bands are not established |
| Weak or absent lysate band | Membrane-associated TNFSF14 may be poorly recovered from the lysate |
💡Expected TNFSF14 appearanceUniProt predicts 26.4 kDa for TNFSF14; no empirical band size is supplied, and glycosylation or isoforms could alter migration, so confirm any candidate band with identity controls.
| Predicted molecular mass | Provides a 26.4 kDa reference, not a measured band position |
| N-linked glycosylation at Asn102 | Could increase apparent mass; the size of any effect is unknown |
| Isoform 1 | Its individual mass and migration are not supplied |
| Isoform 2 | Its size relative to isoform 1 and its migration are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated TNFSF14 may be poorly extracted | Check membrane protein recovery and use a positive control |
| Band higher than expected | Asn102 glycosylation could affect migration, but the shift is unmeasured | Compare treated and untreated samples and confirm band identity |
| Band lower than expected | An isoform or degradation is possible; the supplied features do not establish its size | Check sample integrity and confirm identity with a second antibody |
| Multiple bands | Isoforms 1 and 2 are annotated, but their band positions are unknown | Confirm candidate bands with isoform-aware controls |
| Weak or no signal | Membrane protein recovery may be low | Check extraction and antibody performance with a positive control |
Experiment evidence
Western blot images showing TNFSF14 band
A03516 · TNFSF14
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TNFSF14 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for TNFSF14 in Western blot, you can use adrenal gland tissue, which HPA scores High.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: TNFSF14 has membrane and secreted forms, so signal may differ between tissue lysate and extracellular fractions.
HPA tissue expression evidence for TNFSF14
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 |
|---|---|---|---|---|
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Duodenum | glandular cells | High | Protein (IHC) | HPA → |
| Epididymis | glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TNFSF14 Western Blot Tips
Deeper troubleshooting and optimisation questions for TNFSF14, answered from its protein features.
How should TNFSF14 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.
How do TNFSF14 isoforms affect antibody selection?
Isoforms · Isoform 2 lacks residues 38–73 relative to canonical isoform 1. Check the antibody epitope against this region: an epitope within it may miss isoform 2, while one outside it may detect both. Residue numbers here follow the supplied UniProt canonical sequence.
How can glycosylation be assessed for TNFSF14?
PTM · UniProt lists one N-linked glycosylation site, Asn102, using canonical sequence numbering. Compare untreated and deglycosylated samples to assess whether glycosylation affects migration. A change would need experimental confirmation; the listed site alone does not predict its size.
Does this guide establish induction of TNFSF14?
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 TNFSF14 Western blot?
Transfer · The membrane form is a single-pass type II membrane protein with a predicted mass of 26.4 kDa. Choose transfer conditions suitable for a protein near this size, and check both the membrane and post-transfer gel to confirm recovery. The supplied features do not identify one required transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A03516 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 TNFSF14 Western blot bands be quantified?
Quantitation · Decide whether the measurement should include both isoforms. Isoform 2 lacks canonical residues 38–73, so verify antibody epitope coverage before comparing samples. If multiple bands appear, identify which represent TNFSF14 before combining their signals; an observed reference band size is unavailable.
Why might TNFSF14 migrate away from its predicted 26.4 kDa?
Interpretation · TNFSF14 has an N-linked glycosylation site at Asn102 and two isoforms. Either could affect apparent size, but these features alone do not establish a visible shift. No observed band size is supplied, so compare any measured band with the 26.4 kDa prediction cautiously.
UniProt describes TNFSF14 as a homotrimer, but that does not establish that a trimer survives Western blot sample preparation. Compare bands under relevant sample preparation conditions before assigning a higher band to an oligomer. The listed disulfide bond also does not specify an interchain link.
Consider the two isoforms and the N-linked site at canonical Asn102 when investigating band differences. Isoform 2 lacks canonical residues 38–73. Confirm band identity experimentally before attributing a band to splicing or glycosylation; the supplied features provide no observed band size or confirmed cleavage product.
UniProt describes TNFSF14 as a homotrimer, but that does not establish that a trimer survives Western blot sample preparation. Compare bands under relevant sample preparation conditions before assigning a higher band to an oligomer. The listed disulfide bond also does not specify an interchain link.
Consider the two isoforms and the N-linked site at canonical Asn102 when investigating band differences. Isoform 2 lacks canonical residues 38–73. Confirm band identity experimentally before attributing a band to splicing or glycosylation; the supplied features provide no observed band size or confirmed cleavage product.
Boster reagents
TNFSF14 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
Anti-TNFSF14 Antibody
Cat # A03516
The catalog reports A03516, an anti-TNFSF14 polyclonal antibody with reported Human and Mouse reactivity. Its WB image shows HeLa and Raw264.7 whole cell lysates at 1:500. These examples document the tested contexts, not performance across all samples.
Which to pick: A03516 is the only listed TNFSF14 antibody. It has a WB image using HeLa and Raw264.7 whole cell lysates at 1:500; consider whether those samples and conditions match your experiment.
Source: BosterBio TNFSF14 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O43557.
- Human Protein Atlas. TNFSF14 tissue expression.
- PMC11896206 — target-verified WB comparison
- PMC12590761 — target-verified WB comparison
- PMC4720906 — target-verified WB comparison