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- Table of Contents
SEMA4D / Semaphorin-4D · Western blot design guide
Design a Western Blot for SEMA4D
Real validated SEMA4D Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SEMA4D 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
SEMA4D 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 | ~96.2 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Adipose tissue (IHC candidate; verify WB) +4 more | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated SEMA4D Western Blot Protocols
The M02512-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | SH-SY5Y cell lysate (catalog M02512-1) |
| Gel % | 8–10% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | M02512-1; 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 SEMA4D Western Blot Band Size?
SEMA4D has a predicted full-length mass of 96.2 kDa; glycosylation, signal peptide cleavage, isoforms, and homodimerization could affect migration, but none establishes an observed band size.
| Band near 96.2 kDa | consistent with the predicted full-length precursor, pending band-identity controls |
| Band above 96.2 kDa | N-linked glycosylation may increase apparent size |
| Band slightly below the precursor | signal peptide cleavage may yield a smaller mature protein |
| Band near twice the monomer size | a homodimer may persist under non-reducing conditions |
| Several bands at different positions | isoforms 1 and 2 may contribute if their migration differs |
💡Expected SEMA4D appearanceThe predicted full-length mass is 96.2 kDa; N-linked glycosylation, signal peptide cleavage, and homodimerization may affect migration, but no empirical band size is supplied, so confirm identity with controls.
| Predicted full-length mass | 96.2 kDa provides the sequence-based reference |
| N-linked glycosylation at Asn49, Asn77, Asn139, Asn191, Asn329, Asn379, Asn419, and Asn613 | may increase apparent size or broaden migration if glycan occupancy varies |
| Homodimer formation | may produce a band near twice the monomer size if the dimer persists during electrophoresis |
| Splice isoforms 1 and 2 | may differ in size, but distinct bands are not established |
| Signal peptide at residues 1–21 | cleavage may make the mature protein smaller than the full-length precursor |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane protein may be poorly recovered during extraction | Check membrane enrichment and use a positive-control lysate |
| Band higher than expected | N-linked glycosylation or persistent homodimer | Compare deglycosylated and reducing preparations with an identity control |
| Band lower than expected | Signal peptide cleavage or an isoform may alter size | Check antibody epitope coverage and confirm identity with knockdown |
| Broad smear instead of sharp band | Variable N-linked glycosylation may broaden migration | Compare untreated and deglycosylated samples |
| Multiple bands | Isoforms 1 and 2 or incompletely reduced homodimer may contribute | Compare reducing conditions and verify bands by knockdown |
| Weak or no signal | Incomplete extraction of membrane-associated SEMA4D | Check membrane protein recovery and include a positive control |
Experiment evidence
Western blot images showing SEMA4D band
M02512-1 · SEMA4D
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SEMA4D Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for SEMA4D in Western blot, you can use adipose tissue, which has high HPA expression.
Positive control: Adipose tissue (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: No supplied tissue is undetected, so use siRNA knockdown or a KO line for a clean negative control.
HPA tissue expression evidence for SEMA4D
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 |
|---|---|---|---|---|
| Adipose tissue | adipocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Cervix | squamous epithelial cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Caudate | neuronal cells | Low | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Low | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced SEMA4D Western Blot Tips
Deeper troubleshooting and optimisation questions for SEMA4D, answered from its protein features.
How should SEMA4D 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 could SEMA4D isoforms complicate band assignment?
Isoforms · Two isoforms are listed. In isoform 2, canonical residues 555–738 are replaced by an alternative sequence, and residues 739–862 are missing. Check whether the antibody epitope lies in a shared region before assigning bands to either isoform. The supplied features do not establish their apparent band sizes.
Which glycosylation sites matter when interpreting SEMA4D bands?
PTM · UniProt annotates N-linked sites at residues 49, 77, 139, 191, 329, 379, 419, 613, and 632. These are UniProt coordinates. Glycosylation may affect migration, but the site annotations alone cannot assign a particular band or its apparent mass.
UniProt annotates phosphoserine at residue 833 of the canonical sequence. That residue lies in the 739–862 segment missing from isoform 2. Check which isoform an antibody recognizes before interpreting a phosphorylation signal. Residue 833 uses UniProt numbering, which may differ from paper or antibody numbering; the annotation alone does not predict a visible shift.
UniProt annotates phosphoserine at residue 833 of the canonical sequence. That residue lies in the 739–862 segment missing from isoform 2. Check which isoform an antibody recognizes before interpreting a phosphorylation signal. Residue 833 uses UniProt numbering, which may differ from paper or antibody numbering; the annotation alone does not predict a visible shift.
Does this guide establish induction of SEMA4D?
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 SEMA4D Western blot?
Transfer · SEMA4D is a 96.2 kDa predicted single-pass membrane protein. Use a transfer setup verified to recover proteins around that size, and inspect the membrane and post-transfer gel to assess recovery. The supplied features do not establish one optimal transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M02512-1 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 SEMA4D band intensity be quantified?
Quantitation · Measure the same assigned band across samples within the assay’s linear detection range and normalize for loading. SEMA4D has two isoforms and nine annotated N-linked sites, so define the band or bands included in the measurement before comparing samples. No observed band position was supplied.
Why might SEMA4D migrate differently from 96.2 kDa?
Interpretation · The 96.2 kDa prediction does not account for how SEMA4D migrates on a gel. Its signal peptide spans UniProt residues 1–21, and nine N-linked glycosylation sites are annotated. These features warrant checking the apparent mass, but they do not establish a visible shift; no observed band size was supplied.
SEMA4D is annotated as a homodimer and has eight disulfide bonds. Compare reducing and nonreducing samples and check antibody specificity when investigating higher bands. These features suggest useful comparisons but do not establish that any particular band is a dimer.
SEMA4D is annotated as a homodimer and has eight disulfide bonds. Compare reducing and nonreducing samples and check antibody specificity when investigating higher bands. These features suggest useful comparisons but do not establish that any particular band is a dimer.
Boster reagents
SEMA4D 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-Semaphorin 4D / CD100 Rabbit Monoclonal Antibody
Cat # M02512-1
M02512-1 is a rabbit monoclonal anti-SEMA4D antibody listed with human reactivity. Its Western blot image shows Semaphorin 4D/CD100 expression in SH-SY5Y cell lysate. No other tested contexts are supplied.
Which to pick: M02512-1 is the only listed option. It has a Western blot image from SH-SY5Y cell lysate and is listed as reactive with human samples; performance in other sample contexts is not documented here.
Source: BosterBio SEMA4D gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q92854.
- Human Protein Atlas. SEMA4D tissue expression.
- PMC8988092 — target-verified WB comparison
- PMC8672410 — target-verified WB comparison
- PMC5751932 — target-verified WB comparison