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- Table of Contents
PENK / Proenkephalin-A · Western blot design guide
Design a Western Blot for PENK
Source-linked PENK Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PENK 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
PENK 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 | ~30.8 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +2 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Cleaved | |
| Caveat | Proteolytic processing | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked PENK Western Blot Protocol Options
The A07495-1 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 | A07495-1; 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 PENK Western Blot Band Size?
PENK’s full-length precursor is predicted at 30.8 kDa; cleavage and secretion may affect the blot, but no migration position is demonstrated.
| Band near 30.8 kDa | Consistent with the predicted full-length precursor; identity needs confirmation |
| Band below 30.8 kDa | Could reflect signal-peptide or propeptide cleavage |
| Several smaller bands | Could reflect processing at paired basic residues |
| Little or no band in whole-cell lysate | PENK localizes to secretory vesicles and is secreted |
💡Expected PENK appearanceThe full-length precursor has a predicted mass of 30.8 kDa, but no empirical band size is supplied; confirm any candidate band with appropriate identity controls.
| Predicted full-length mass | Provides a 30.8 kDa reference for the precursor, not a measured migration position |
| Signal peptide at residues 1–24 | Its cleavage can yield a smaller product than the full-length precursor |
| Propeptide at residues 196–207 | Its cleavage can yield a smaller processed species |
| Cleavage at paired basic residues | Processing can produce smaller fragments of unspecified masses |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | PENK is present in secretory vesicles and is secreted | Check a secretory-vesicle-enriched fraction or conditioned medium |
| Band lower than expected | Signal-peptide or propeptide cleavage may reduce size | Compare precursor-enriched and secreted samples with antibodies to mapped regions |
| Multiple bands | The precursor and processed products may coexist | Compare sample fractions and confirm bands with antibodies to distinct PENK regions |
| Weak or no signal | Processing may remove the region recognized by the antibody | Check antibody epitope coverage and probe a precursor-enriched fraction |
| Fragments below expected size | Cleavage at paired basic residues can generate smaller products | Confirm fragment identity with antibodies to mapped precursor regions |
Experiment evidence
Western blot images showing PENK band
A07495-1 · PENK
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PENK Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PENK in Western blot, you can use adrenal gland tissue, which has high HPA expression.
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 alongside the samples.
⚠️Feasibility: PENK is secreted, so whole-cell lysates may give a weak signal; consider conditioned medium.
HPA tissue expression evidence for PENK
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 | medullary cells | High | Protein (IHC) | HPA → |
| Caudate | neuronal cells | Medium | Protein (IHC) | HPA → |
| Testis | spermatogonia 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 → |
| Appendix | endocrine cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PENK Western Blot Tips
Deeper troubleshooting and optimisation questions for PENK, answered from its protein features.
How can PENK processing affect which bands appear?
Band shift · PENK has a propeptide at residues 196–207 and is annotated for cleavage at pairs of basic residues. An antibody may detect the precursor, a processed product, or both, depending on its epitope. Check which part of PENK the antibody recognizes before assigning bands.
Could annotated PENK isoforms explain multiple bands?
Isoforms · Only one isoform is supplied, with no alternative sequence. Do not assign multiple bands to annotated isoforms on this evidence. Consider the listed signal peptide and processing features when evaluating band identities.
Should phosphorylation change PENK band migration?
PTM · UniProt lists phosphoserine at position 251 in its 267-residue sequence. Use UniProt numbering when comparing this site with antibody or paper coordinates. The modification is relevant to epitope choice, but its presence alone does not establish a visible band shift.
Does this guide establish induction of PENK?
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 PENK?
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 A07495-1 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 PENK Western blot signals be quantified?
Quantitation · Define the band or bands being measured and confirm that the antibody recognizes the intended PENK region. Its signal peptide and processing annotations mean precursor and peptide products should not automatically be combined into one quantity.
Why might a PENK band differ from the predicted 30.8 kDa?
Interpretation · The 30.8 kDa prediction refers to the full 267-residue sequence. PENK has a signal peptide at residues 1–24 and features associated with peptide processing, so a detected product may differ in size. No observed band is supplied; these features alone cannot establish the cause of a particular shift.
PENK is annotated in the lumen of secretory vesicles and chromaffin granules and as secreted. Choose and document the sample fraction accordingly; cellular and secreted samples may contain different PENK products.
Compare antibody epitope coverage with PENK's signal peptide at residues 1–24, propeptide at 196–207, and basic-residue cleavage annotation. Its three disulfide bonds may also make reducing conditions relevant to interpretation. Without band-specific evidence, these features identify possibilities, not the identity of an unexpected band.
PENK is annotated in the lumen of secretory vesicles and chromaffin granules and as secreted. Choose and document the sample fraction accordingly; cellular and secreted samples may contain different PENK products.
Compare antibody epitope coverage with PENK's signal peptide at residues 1–24, propeptide at 196–207, and basic-residue cleavage annotation. Its three disulfide bonds may also make reducing conditions relevant to interpretation. Without band-specific evidence, these features identify possibilities, not the identity of an unexpected band.
Boster reagents
PENK 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-Proenkephalin-A PENK Antibody
Cat # A07495-1
A07495-1 is a polyclonal anti-PENK antibody listed for Western blot, with a WB image and reported human, mouse, and rat reactivity. The supplied listing gives only a brief image cue, limiting interpretation of the blot evidence.
Which to pick: A07495-1 is the only listed PENK antibody. It has a WB image and reported reactivity for human, mouse, and rat; review the full blot details for your sample and conditions.
Source: BosterBio PENK gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P01210.
- Human Protein Atlas. PENK tissue expression.
- PMC7793737 — target-verified WB comparison