This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
SEMA7A / Semaphorin-7A · Western blot design guide
Design a Western Blot for SEMA7A
Source-linked SEMA7A Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SEMA7A 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
SEMA7A Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~74.8 kDa | |
| Observed band | ≈75 kDa | |
| Gel | 5–20% (catalog A03832-2) | |
| Positive control | Appendix (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 + Methylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked SEMA7A Western Blot Protocol Options
The A03832-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human U251 (catalog A03832-2) |
| Gel % | 5–20% (catalog A03832-2) |
| Load | 30 ug; reducing conditions (catalog A03832-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A03832-2) |
| Membrane | nitrocellulose membrane (catalog A03832-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A03832-2) |
| Primary antibody | A03832-2 · 0.5 μg/mL (catalog A03832-2) |
| Primary incubation | overnight at 4°C (catalog A03832-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A03832-2) |
| Secondary incubation | 1.5 hour at RT (catalog A03832-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A03832-2) |
| Detection | ECL (catalog A03832-2) |
Section 2
What Is the Expected SEMA7A Western Blot Band Size?
SEMA7A is 74.8 kDa predicted and approximately 75 kDa observed; the cause of their small difference is not established.
| Band near 75 kDa | matches the empirical SEMA7A band in reducing whole-cell lysate |
| Band above 75 kDa | could reflect N-linked glycosylation; the size effect is unquantified |
| Band below 75 kDa | could reflect signal-peptide or propeptide cleavage; migration is unquantified |
| Multiple bands | could reflect isoforms 1 and 2 if they migrate differently |
| Weak band in soluble fraction | SEMA7A is GPI-anchored at the cell membrane |
💡Expected SEMA7A appearanceSEMA7A has a predicted mass of 74.8 kDa and an observed band near 75 kDa in reducing whole-cell lysate; confirm identity with an appropriate antibody and positive control.
| Predicted SEMA7A mass | 74.8 kDa predicted, with an empirical band near 75 kDa |
| N-linked glycosylation at Asn105, Asn157, Asn258, Asn330, and Asn602 | may alter apparent mass; the extent is not supplied |
| Signal peptide at residues 1–44 | cleavage makes the mature protein smaller than its precursor |
| Propeptide at residues 649–666 | cleavage makes the processed protein smaller than its precursor |
| Isoforms 1 and 2 | may differ in size; their individual masses are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | GPI-anchored SEMA7A may be poorly represented in the sampled fraction | Check a membrane-enriched fraction and a positive control |
| Band higher than expected | N-linked glycosylation may affect migration | Compare treated and untreated samples with an identity control |
| Band lower than expected | Signal-peptide or propeptide cleavage may reduce size | Check antibody epitope coverage and confirm band identity |
| Broad smear instead of sharp band | Heterogeneous N-linked glycosylation is possible | Compare glycosidase-treated and untreated samples |
| Multiple bands | Isoforms 1 and 2 or different processing states are possible | Confirm each band with an independent SEMA7A antibody |
| Weak or no signal | Membrane localization may limit recovery in the prepared fraction | Check membrane recovery and include a positive control |
Experiment evidence
Western blot images showing SEMA7A band
A03832-2 · SEMA7A
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SEMA7A Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for SEMA7A in Western blot, you can use appendix tissue, which HPA reports as positive.
Positive control: Appendix (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: SEMA7A is GPI-anchored at the cell surface and can be extracellular, so whole-cell lysate signal may vary.
HPA tissue expression evidence for SEMA7A
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 |
|---|---|---|---|---|
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | Medium | Protein (IHC) | HPA → |
| Colon | glandular cells | Medium | Protein (IHC) | HPA → |
| Hippocampus | neuronal cells | Medium | 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 → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced SEMA7A Western Blot Tips
Deeper troubleshooting and optimisation questions for SEMA7A, answered from its protein features.
How should SEMA7A 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.
Could SEMA7A isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. Isoform 2 lacks residues 111–124 relative to the canonical sequence. That difference could affect band interpretation, but the supplied features do not establish whether the isoforms resolve as separate bands. Check which isoform your sample and antibody can detect.
Which SEMA7A modifications should I consider?
PTM · UniProt lists N-linked glycosylation at Asn105, Asn157, Asn258, Asn330, and Asn602, plus asymmetric dimethylarginine at Arg135. These are canonical UniProt coordinates; antibody or paper numbering may differ. The annotations alone do not establish a visible shift.
Does this guide establish induction of SEMA7A?
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 SEMA7A Western blot?
Transfer · The supplied features do not specify a transfer method. SEMA7A is a GPI-anchored protein on the extracellular side of the cell membrane, with an observed band near 75 kDa. Check whether your chosen transfer conditions retain that band on the membrane.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A03832-2 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 I quantify SEMA7A bands?
Quantitation · Define consistently whether you measure the approximately 75 kDa band alone or additional resolved species. UniProt lists two isoforms and five N-linked glycosylation sites, so record which bands you include across samples. The supplied features do not identify which species contribute to any particular band.
Why does SEMA7A run near 75 kDa?
Interpretation · The observed band near 75 kDa is close to the 74.8 kDa predicted mass. UniProt also annotates a signal peptide at residues 1–44, a propeptide at 649–666, and five N-linked glycosylation sites. These features can affect a processed protein’s mass, but their presence alone does not explain a specific band position.
Compare them with the approximately 75 kDa observed band, then consider isoform 2’s deletion of canonical residues 111–124, the signal peptide at 1–44, the propeptide at 649–666, and the listed modifications. None of these annotations alone identifies an unexpected band or proves a visible shift.
UniProt places SEMA7A on the extracellular side of the cell membrane and identifies it as GPI-anchored. When comparing samples, keep the fraction and preparation consistent; differences in membrane protein recovery could affect the measured signal.
Compare them with the approximately 75 kDa observed band, then consider isoform 2’s deletion of canonical residues 111–124, the signal peptide at 1–44, the propeptide at 649–666, and the listed modifications. None of these annotations alone identifies an unexpected band or proves a visible shift.
UniProt places SEMA7A on the extracellular side of the cell membrane and identifies it as GPI-anchored. When comparing samples, keep the fraction and preparation consistent; differences in membrane protein recovery could affect the measured signal.
Boster reagents
SEMA7A 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 7a/SEMA7A Antibody Picoband®
Cat # A03832-2
The catalog reports one anti-SEMA7A antibody, A03832-2, with reported human reactivity. Its Western blot image shows an approximately 75 kDa band in human U251 whole cell lysate under reducing conditions. The supplied evidence covers this tested sample only.
Which to pick: A03832-2 is the only listed option and has a Western blot image from human U251 lysate. Use its reported conditions as a starting point; performance in other samples is not established here.
Source: BosterBio SEMA7A gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O75326.
- Human Protein Atlas. SEMA7A tissue expression.
- PMC12634057 — target-verified WB comparison
- PMC12363069 — target-verified WB comparison
- PMC10978839 — target-verified WB comparison