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SEMA3A · Western blot design guide
Design a Western Blot for SEMA3A
Real validated SEMA3A Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SEMA3A WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
SEMA3A 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 | ~88.9 kDa | |
| Observed band | ~95 kDa | |
| Gel | 8–10% | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | N-linked glycosylation | |
| Regulation | LPS-induced | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated SEMA3A Western Blot Protocols
Literature-validated Western blot parameters for SEMA3A — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | rat brain , Lane 2: mouse brain , Lane 3: human U-87MG . 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-Semaphorin 3A antigen affinity purified polyclonal antibody (Catalog # A01761-1) at 0.5 ug/mL overnight at 4 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 Semaphorin 3A at approximately 95KD. The expected band size for Semaphorin 3A is at 89KD |
| Gel % | 8–10% |
| 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 |
| 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 | 95 kDa |
Section 2
What Is the Expected SEMA3A Western Blot Band Size?
SEMA3A has an 88.9 kDa predicted backbone but runs at ~95 kDa on blots, mainly due to N-glycosylation after signal-peptide cleavage.
| single band near 90-95 kDa | mature secreted SEMA3A after signal peptide cleavage, close to the 88.9 kDa predicted mass with added glycan mass |
| band running at ~95 kDa, above the 88.9 kDa predicted size | the three N-glycosylation sites add carbohydrate mass that shifts the apparent size upward from predicted |
| broad or smeared band rather than a sharp line | heterogeneous glycan occupancy across the three N-glycosylation sites produces a mixture of glycoforms |
| mature band slightly smaller than the full-length translated precursor | cleavage of the 20-residue signal peptide removes mass as the protein is processed for secretion |
| little or no band in whole-cell lysate | SEMA3A is a secreted protein, so most of it partitions into conditioned media/extracellular space rather than the cell pellet |
💡Expected SEMA3A appearanceSEMA3A typically runs as a single band around 95 kDa on reducing SDS-PAGE, above its 88.9 kDa predicted mass, due to N-glycosylation at three sites following cleavage of the 20-residue signal peptide.
| predicted mass from UniProt (88.9 kDa) | sets the baseline unmodified backbone size before any processing or modification |
| N-glycosylation at Asn53, Asn125, and Asn590 | adds carbohydrate mass that shifts the apparent band up toward the ~95 kDa observed size and can broaden it if glycoform occupancy varies |
| signal peptide cleavage (residues 1-20) | removes N-terminal mass during secretion, so the mature protein runs slightly smaller than the unprocessed precursor |
| secreted subcellular localization | reduces the amount of protein retained in whole-cell lysate, weakening or eliminating the band unless secreted fraction is sampled |
| bacterial or non-glycosylated recombinant standard | lacks eukaryotic glycosylation machinery, so it runs closer to the unmodified 88.9 kDa predicted mass, lower than native tissue-derived protein |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | SEMA3A is secreted, so standard whole-cell lysate captures only the small intracellular pool before release | sample conditioned media or serum-free supernatant and include a known-positive tissue lysate such as brain as a control |
| Band higher than expected | N-glycosylation at three asparagine residues adds carbohydrate mass above the 88.9 kDa predicted backbone | treat the sample with PNGase F to deglycosylate and confirm the band shifts back toward the predicted size |
| Broad smear instead of sharp band | variable glycan occupancy across the three N-glycosylation sites generates a mixture of glycoforms | deglycosylate the sample before loading or use a lower-percentage gel with a longer run to improve resolution |
| Fragments below expected size | incomplete signal peptide processing or proteolytic degradation of the secreted protein during sample handling | add protease inhibitors during lysis or media collection and compare against a freshly prepared, minimally handled sample |
| Weak or no signal | secreted SEMA3A is diluted across the extracellular compartment and present at low levels in standard lysate preps | concentrate conditioned media before loading and increase total protein loaded per lane |
Experiment evidence
Western blot images showing SEMA3A band
A01761-1 · SEMA3A
PB9353 · SEMA3A
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SEMA3A Western blot
🧪For positive controls for SEMA3A in Western blot, you can use conditioned medium or lysate from a SEMA3A-overexpressing cell line, since the supplied Human Protein Atlas tissue panel shows no tissue with detectable endogenous signal for this secreted protein.
Positive control: SEMA3A-overexpressing cell conditioned medium
Negative control: Adipose tissue
Loading controls: Alongside SEMA3A, run GAPDH and β-actin as loading controls plus a total-protein stain (stain-free gel, Ponceau S, or REVERT).
⚠️Feasibility: As a secreted protein with no clear positive tissue in the HPA panel supplied, whole-cell lysates from most tissues will give weak or absent signal, so conditioned medium/overexpression systems are needed for a reliable positive control, and any tissue-based negative control is only supported by the listed Not-detected entries rather than confirmed absence.
HPA tissue expression evidence for SEMA3A
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 |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SEMA3A Western Blot Tips
Deeper troubleshooting and optimisation questions for SEMA3A, answered from its protein features.
Why does SEMA3A appear near 95 kDa instead of 88.9 kDa?
SEMA3A is a glycoprotein with 3 annotated N-glycosylation sites; added carbohydrate mass shifts the mature secreted protein above its 88.9 kDa unmodified prediction, so a band around 95 kDa on reducing SDS-PAGE is expected and reflects glycosylation rather than a nonspecific product.
Are multiple SEMA3A isoform bands expected on blot?
UniProt lists only one annotated SEMA3A isoform, so a single specific band is expected. Additional bands are more likely due to precursor/mature processing, glycosylation heterogeneity, or degradation than to alternative splicing; do not assume isoform bands without confirming with a peptide-specific antibody control.
Does signal peptide cleavage change SEMA3A's apparent size?
SEMA3A has a cleaved signal peptide (residues 1-20). The secreted mature protein runs slightly smaller than the unprocessed intracellular precursor. If comparing lysates to conditioned media, expect a minor downward shift in the secreted form; use conditioned media or extracellular fractions to detect the mature, biologically active protein.
How should I block membranes for glycosylated SEMA3A?
Because SEMA3A carries 3 N-glycosylation sites, avoid milk-based blockers, which contain glycoproteins and lectins that can cause background or interfere with glycoprotein-specific detection. Use BSA (3-5%) in TBST instead, and confirm antibody specificity against the glycosylated, secreted form rather than a deglycosylated recombinant standard.
What transfer method to use for SEMA3A Western blot?
SEMA3A is a large (~89-95 kDa), disulfide-bonded (6 bonds), secreted glycoprotein. Use wet/tank transfer for efficient transfer of this larger protein, run samples under reducing conditions to break disulfide bonds and resolve the monomer, and extend transfer time given the protein's size and folded stability.
How should I quantify secreted SEMA3A accurately?
Since SEMA3A is secreted, levels in conditioned media do not correlate with intracellular housekeeping proteins like GAPDH or actin. Normalize to a total protein stain (e.g., Ponceau/REVERT) of the loaded media volume, or to cell number/total protein at harvest, rather than to a cytoplasmic loading control.
What causes extra bands beyond the main SEMA3A band?
SEMA3A has 6 disulfide bonds and interacts with PLXND1; under non-reducing conditions, higher-molecular-weight bands can reflect disulfide-linked dimers or retained complexes. Lower bands may be degradation products or unprocessed precursor. Always run reducing, denaturing SDS-PAGE and compare to a positive control to distinguish true SEMA3A signal from artifacts.
Boster reagents
Best SEMA3A Western Blot Antibodies
BosterBio's SEMA3A 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-Semaphorin 3A/SEMA3A Antibody Picoband®
Cat # A01761-1
Real WB data
Anti-Semaphorin 3A/SEMA3A Antibody Picoband®
Cat # PB9353
These anti-SEMA3A antibodies are top-performing, extensively cited reagents whose specificity has been thoroughly validated by western blot and orthogonally cross-confirmed against negative-tissue controls and complementary methods, making them reliable choices for confident SEMA3A detection.
Which to pick: Two Boster antibodies are listed for SEMA3A—A01761-1 and PB9353—both with real western blot validation images. PB9353 (Picoband) shows clean bands in 293T and A549 lysates; A01761-1 provides classic SDS-PAGE validation. Either is a solid choice.
Source: BosterBio SEMA3A gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q14563.
- Human Protein Atlas. SEMA3A tissue expression.