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PDE5A / cGMP-specific 3',5'-cyclic phosphodiesterase · Western blot design guide
Design a Western Blot for PDE5A
Source-linked PDE5A Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PDE5A 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
PDE5A 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 | ~100 kDa | |
| Observed band | ~100 kDa | |
| Gel | 8% (catalog A02490-2) | |
| Positive control | Colon (IHC candidate; verify WB) +4 more | |
| Negative control | Vagina (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked PDE5A Western Blot Protocol Options
The A02490-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | rat lung, mouse lung (catalog A02490-2) |
| Gel % | 8% (catalog A02490-2) |
| Load | 30 ug; reducing conditions (catalog A02490-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A02490-2) |
| Membrane | nitrocellulose membrane (catalog A02490-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A02490-2) |
| Primary antibody | A02490-2 · 0.5 μg/mL (catalog A02490-2) |
| Primary incubation | overnight at 4°C (catalog A02490-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A02490-2) |
| Secondary incubation | 1.5 hour at RT (catalog A02490-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A02490-2) |
| Detection | ECL (catalog A02490-2) |
Section 2
What Is the Expected PDE5A Western Blot Band Size?
PDE5A is predicted at 100 kDa and observed at ~100 kDa; annotated isoforms do not establish additional band sizes.
| Band at ~100 kDa | Matches the observed PDE5A band and UniProt predicted mass |
| Single sharp band near 100 kDa | PDE5A1 and PDE5A2 need not resolve as separate bands |
| Two bands near 100 kDa | Could reflect PDE5A1 and PDE5A2, pending identity controls |
| No visible mobility shift near 100 kDa | Phosphorylation at Ser102 need not produce a resolvable shift |
💡Expected PDE5A appearanceUniProt predicts 100 kDa, and antibody blots show a band at ~100 kDa; use antibody specificity controls to confirm its identity.
| UniProt predicted mass | Predicts 100 kDa, matching the observed ~100 kDa band |
| PDE5A1 splice isoform | May differ in size from PDE5A2; its individual migration is unknown |
| PDE5A2 splice isoform | May differ in size from PDE5A1; its individual migration is unknown |
| Alternative splicing | Can affect apparent size, but distinct resolvable bands are not established |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | Its identity is uncertain because no higher migrating PDE5A form is established here | Check the ~100 kDa reference band and antibody specificity |
| Band lower than expected | An isoform difference is possible, but individual isoform migration is unknown | Confirm identity with an independent PDE5A antibody or depletion control |
| Multiple bands | PDE5A1 and PDE5A2 are annotated isoforms, but separate bands are unproven | Compare bands with an isoform specific assay or depletion control |
| Weak or no signal | PDE5A abundance or assay sensitivity may be insufficient | Include a validated positive lysate and check loading and transfer |
| Fragments below expected size | Proteolysis or nonspecific binding may produce smaller bands | Prepare fresh lysate with protease inhibitors and confirm band identity |
Experiment evidence
Western blot images showing PDE5A band
A02490-2 · PDE5A
A02490-2 · PDE5A
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PDE5A Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PDE5A in Western blot, you can use colon tissue, which has high HPA expression.
Positive control: Colon (IHC candidate; verify WB)
Negative control: Vagina (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: A tissue contrast is feasible because HPA reports high expression in colon and no detection in vagina.
HPA tissue expression evidence for PDE5A
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 |
|---|---|---|---|---|
| Colon | peripheral nerve/ganglion | High | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Vagina | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | Low | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Low | Protein (IHC) | HPA → |
| Seminal vesicle | glandular cells | Low | Protein (IHC) | HPA → |
| Tonsil | germinal center cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced PDE5A Western Blot Tips
Deeper troubleshooting and optimisation questions for PDE5A, answered from its protein features.
What band size should I expect for PDE5A?
Band shift · PDE5A has a predicted mass of about 100 kDa, matching the supplied observed band near 100 kDa. Apparent migration can vary, so size alone does not establish band identity.
Consider the two listed isoforms and whether the antibody recognizes their differing N termini. UniProt also lists phosphoserine 102, but its presence alone does not establish a shifted band. The supplied features list no glycosylation sites, signal peptide, or propeptide to support those explanations.
Consider the two listed isoforms and whether the antibody recognizes their differing N termini. UniProt also lists phosphoserine 102, but its presence alone does not establish a shifted band. The supplied features list no glycosylation sites, signal peptide, or propeptide to support those explanations.
How does antibody epitope location affect isoform detection?
Isoforms · An antibody recognizing the differing N-terminal region may distinguish PDE5A1 from PDE5A2. An epitope in their shared sequence may detect both. Check the antibody's stated epitope before interpreting isoform bands.
PDE5A1 and PDE5A2 differ at the N terminus. If an antibody detects both and their bands overlap, report the signal as combined PDE5A. Quantify isoforms separately only when the bands and antibody specificity support that assignment.
PDE5A1 and PDE5A2 differ at the N terminus. If an antibody detects both and their bands overlap, report the signal as combined PDE5A. Quantify isoforms separately only when the bands and antibody specificity support that assignment.
Does phosphorylation explain a shifted PDE5A band?
PTM · UniProt lists phosphoserine at position 102 of its canonical sequence. This feature alone does not demonstrate a visible shift or explain a mass difference. Keep the UniProt numbering convention explicit when comparing the site with antibody or paper labels.
Does this guide establish induction of PDE5A?
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.
How should transfer be checked for PDE5A?
Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A02490-2 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 PDE5A be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Could PDE5A1 and PDE5A2 appear as different bands?
Interpretation · PDE5A2 replaces PDE5A1 residues 1–49 with a seven-residue sequence, making it shorter. The features do not establish whether the isoforms resolve as separate bands on a blot.
Boster reagents
PDE5A 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-PDE5A Antibody Picoband®
Cat # A02490-2
A02490-2 is listed for human, mouse, and rat PDE5A. Its Western blot image shows an approximately 100 kDa band in rat and mouse lung lysates under reducing conditions. No human Western blot example or publication evidence is supplied.
Which to pick: A02490-2 is the only listed option. It has a Western blot image for rat and mouse lung lysates; human reactivity is listed, but no human Western blot example is provided.
Source: BosterBio PDE5A gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O76074.
- Human Protein Atlas. PDE5A tissue expression.
- PMC13098669 — target-verified WB comparison
- PMC12279358 — target-verified WB comparison
- PMC6350610 — target-verified WB comparison