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- Table of Contents
PIP / Prolactin-inducible protein · Western blot design guide
Design a Western Blot for PIP
Real validated PIP Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PIP 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
PIP 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 | ~16.6 kDa | |
| Gel | 15% (standard starting point) | |
| Positive control | Salivary gland (IHC candidate; verify WB) +1 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated PIP Western Blot Protocols
The M04543-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | T47D cell lysate (catalog M04543-2) |
| Gel % | 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 | 0.2 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | M04543-2; 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 PIP Western Blot Band Size?
PIP has a predicted 16.6 kDa precursor; signal peptide cleavage and Asn105 glycosylation may affect migration, but no empirical band or feature-specific shift is established.
| Band near 16.6 kDa | May represent the full-length precursor; confirm band identity |
| Band below the precursor position | May reflect removal of the 1–28 signal peptide |
| Band above an unglycosylated form | Could reflect N-linked glycosylation at Asn105; a visible shift is unproven |
| Little or no band in whole-cell lysate | PIP is secreted and may be more abundant in conditioned medium |
💡Expected PIP appearanceThe predicted 16.6 kDa mass is for the precursor; signal peptide removal and possible Asn105 glycosylation may affect migration, but no empirical band size is supplied, so confirm identity with controls.
| Predicted precursor mass | 16.6 kDa before signal peptide removal or modification |
| Signal peptide at residues 1–28 | cleavage produces a smaller mature chain than the precursor |
| N-linked glycosylation site at Asn105 | glycosylation could increase apparent size; the shift is not established |
| Unglycosylated Asn105 form | could migrate below a glycosylated form if both are present |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | PIP is secreted | check conditioned medium and a positive control |
| Band higher than expected | possible Asn105 glycosylation; the size effect is unverified | compare untreated and deglycosylated samples with a positive control |
| Band lower than expected | signal peptide removal may yield a smaller mature chain | compare lysate and conditioned medium and verify antibody specificity |
| Multiple bands | precursor and mature forms or differing Asn105 glycosylation may be present | compare sample fractions and use a specificity control |
| Weak or no signal | secreted PIP may be scarce in the sampled lysate | test conditioned medium and a known positive sample |
Experiment evidence
Western blot images showing PIP band
M04543-2 · PIP
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PIP Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PIP in Western blot, you can use salivary gland tissue.
Positive control: Salivary 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: PIP is secreted, so conditioned medium may yield a stronger signal than whole-cell lysate.
HPA tissue expression evidence for PIP
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 |
|---|---|---|---|---|
| Salivary gland | glandular cells | High | Protein (IHC) | HPA → |
| Breast | glandular 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 → |
| 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 → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PIP Western Blot Tips
Deeper troubleshooting and optimisation questions for PIP, answered from its protein features.
How should PIP 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 PIP isoforms explain multiple bands?
Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. It therefore provides no isoform-based explanation for multiple bands. Compare bands with the listed signal-peptide processing and modification features before assigning identities.
Where are PIP's listed modifications?
PTM · In UniProt numbering, residue 29 has pyrrolidone carboxylic acid and Asn105 is an N-linked glycosylation site. Residue 29 follows the 1–28 signal peptide. Check the numbering convention before comparing these sites with an antibody datasheet or paper.
Does this guide establish induction of PIP?
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 PIP Western blot?
Transfer · PIP is predicted to be 16.6 kDa before removal of its 1–28 signal peptide. Use transfer conditions suited to a small protein, such as a 0.2 µm membrane, and check the post-transfer gel for protein left behind.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M04543-2 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 secreted PIP be quantified?
Quantitation · PIP is annotated as secreted. Compare the same sample fraction across conditions, and keep conditioned-medium collection and loading consistent. A lysate measurement alone may miss PIP released into the medium.
Why might PIP migrate differently from its predicted 16.6 kDa mass?
Interpretation · The 16.6 kDa prediction includes the 1–28 signal peptide, which is removed from secreted PIP. UniProt also lists N-linked glycosylation at Asn105 and pyrrolidone carboxylic acid at residue 29. These features are relevant when interpreting migration, but they do not establish a visible shift or its size.
Check whether bands differ between conditioned medium and lysate, since PIP is secreted and has a 1–28 signal peptide. Its listed Asn105 glycosylation and two disulfide bonds may also affect migration under different sample conditions. The features alone cannot identify an unexpected band.
UniProt lists two disulfide bonds in PIP. Compare matched reduced and nonreduced samples if migration differs between preparations. A mobility change under those conditions would be consistent with disulfide-dependent structure, but would not by itself establish band identity.
Check whether bands differ between conditioned medium and lysate, since PIP is secreted and has a 1–28 signal peptide. Its listed Asn105 glycosylation and two disulfide bonds may also affect migration under different sample conditions. The features alone cannot identify an unexpected band.
UniProt lists two disulfide bonds in PIP. Compare matched reduced and nonreduced samples if migration differs between preparations. A mobility change under those conditions would be consistent with disulfide-dependent structure, but would not by itself establish band identity.
Boster reagents
PIP 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-GCDFP 15 Rabbit Monoclonal Antibody
Cat # M04543-2
The listed anti-PIP antibody, M04543-2, is a rabbit monoclonal with stated human reactivity. Its Western blot image shows GCDFP 15 expression in T47D cell lysate. No broader sample or species validation is supplied.
Which to pick: M04543-2 is the only listed option and has a Western blot image from T47D cell lysate. Consider it for human PIP Western blotting; performance in other samples is not established by the supplied evidence.
Source: BosterBio PIP gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P12273.
- Human Protein Atlas. PIP tissue expression.
- PMC6360109 — target-verified WB comparison
- PMC3670933 — target-verified WB comparison
- PMC3376385 — target-verified WB comparison