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- Table of Contents
PEG3 / Paternally-expressed gene 3 protein · Western blot design guide
Design a Western Blot for PEG3
Real validated PEG3 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PEG3 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
PEG3 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 | ~180.8 kDa | |
| Observed band | ~200 kDa | |
| Gel | 5–20% (catalog A02541-2) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | — | |
| Caveat | Observed above expected | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 4 isoform(s) |
Section 1
Real Curated PEG3 Western Blot Protocols
The A02541-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human HepG2 (catalog A02541-2) |
| Gel % | 5–20% (catalog A02541-2) |
| Load | 30 ug; reducing conditions (catalog A02541-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A02541-2) |
| Membrane | nitrocellulose membrane (catalog A02541-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A02541-2) |
| Primary antibody | A02541-2 · 0.5 μg/mL (catalog A02541-2) |
| Primary incubation | overnight at 4°C (catalog A02541-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A02541-2) |
| Secondary incubation | 1.5 hour at RT (catalog A02541-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A02541-2) |
| Detection | ECL (catalog A02541-2) |
Section 2
What Is the Expected PEG3 Western Blot Band Size?
PEG3 is predicted at 180.8 kDa and observed near 200 kDa in reducing HepG2 lysate; the cause of the difference is not established.
| Band near 200 kDa | Empirical PEG3 band in reducing HepG2 lysate; confirm identity with antibody controls |
| Band near 181 kDa | Near the 180.8 kDa sequence-predicted mass; identity requires confirmation |
| Band near twice the monomer size | Could reflect a PEG3 homodimer if it survives sample preparation |
| Additional discrete bands | Could reflect isoforms 1, 2, 3, or 4; their separation is unestablished |
💡Expected PEG3 appearancePEG3 has a predicted mass of 180.8 kDa, while a reducing HepG2 blot shows a band near 200 kDa; the difference is unexplained, so confirm band identity with antibody controls.
| Predicted sequence mass | 180.8 kDa is the calculated mass; the empirical band is near 200 kDa for an unestablished reason |
| PEG3 homodimer | Could appear near twice the monomer size if the complex survives sample preparation |
| Isoforms 1 and 2 | Alternative splicing could alter apparent size; individual masses and separation are unknown |
| Isoforms 3 and 4 | Alternative splicing could alter apparent size; individual masses and separation are unknown |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | PEG3 abundance in the tested lysate is unknown | Include the HepG2 lysate used for the reported 200 kDa band as a positive control |
| Band higher than expected | The observed 200 kDa band exceeds the predicted 180.8 kDa mass for an unknown reason; a much higher band could involve homodimerization | Compare reducing and nonreducing samples and confirm antibody specificity |
| Band lower than expected | A PEG3 splice isoform is possible, but isoform masses are unavailable | Confirm identity with a second PEG3 antibody or an isoform-specific assay |
| Multiple bands | PEG3 has isoforms 1, 2, 3, and 4, but distinct band positions are unestablished | Check band identity with a second antibody or isoform-specific assay |
| Weak or no signal | PEG3 may be scarce in the sample or transfer of this large protein may be inefficient | Use a HepG2 positive control and check transfer near 200 kDa |
Experiment evidence
Western blot images showing PEG3 band
A02541-2 · PEG3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PEG3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PEG3 in Western blot, you can use adrenal gland tissue, which HPA scores High.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside.
⚠️Feasibility: No HPA tissue is Not detected, so use siRNA knockdown or a KO line for a clean negative control.
HPA tissue expression evidence for PEG3
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 | glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Cerebellum | cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | glial cells | High | Protein (IHC) | HPA → |
| Cervix | glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Lymph node | germinal center cells | Low | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | adipocytes | Medium | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced PEG3 Western Blot Tips
Deeper troubleshooting and optimisation questions for PEG3, answered from its protein features.
How should PEG3 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 PEG3 isoforms produce different bands?
Isoforms · Yes. UniProt lists four isoforms. Isoforms 2 and 4 lack residues 1–126; isoform 3 lacks residues 258–287 and 551–1588. These sequence differences could produce bands of different sizes. Check which isoforms your sample and antibody can detect before assigning a band.
Match the antibody’s binding region to the isoforms of interest. A region within residues 1–126 will be absent from isoforms 2 and 4; regions within 258–287 or 551–1588 will be absent from isoform 3. These coordinates refer to the supplied UniProt canonical sequence.
Match the antibody’s binding region to the isoforms of interest. A region within residues 1–126 will be absent from isoforms 2 and 4; regions within 258–287 or 551–1588 will be absent from isoform 3. These coordinates refer to the supplied UniProt canonical sequence.
Do the listed features support a PEG3 modification-induced band shift?
PTM · No modified residues or glycosylation sites are listed in the supplied features. Those entries provide no basis to attribute the approximately 200 kDa band to a specific modification. They also do not establish that PEG3 cannot be modified.
Does this guide establish induction of PEG3?
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 PEG3 Western blot?
Transfer · PEG3’s canonical sequence is 1,588 residues, with a predicted mass of 180.8 kDa. Use a transfer setup suitable for a large protein, then check the post-transfer gel for remaining PEG3-sized material. Adjust transfer conditions if substantial protein remains.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A02541-2 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 can PEG3 bands be quantified reliably?
Quantitation · Quantify the same validated PEG3 band across samples, keeping exposures within the linear detection range. If multiple bands appear, resolve which isoform or species each represents before combining their signals. PEG3 is listed in both the nucleus and cytoplasm, so compare equivalent sample fractions.
Why might PEG3 appear near 200 kDa instead of 180.8 kDa?
Interpretation · The reported apparent band is about 200 kDa, while the predicted mass is 180.8 kDa. The supplied features do not establish the cause of that difference. Compare the band with an appropriate molecular weight ladder and confirm its identity before assigning a mechanism.
Consider the annotated isoforms first. Isoform 3 lacks residues 258–287 and 551–1588; isoforms 2 and 4 lack residues 1–126. Compare band detection with the antibody’s binding region and the expected isoform sizes. The features alone cannot identify an observed smaller band.
UniProt describes PEG3 as a homodimer, but that annotation alone does not establish that a dimer survives Western blot preparation or causes a particular band. If a higher band appears, assess its identity experimentally before assigning it to a dimer.
Consider the annotated isoforms first. Isoform 3 lacks residues 258–287 and 551–1588; isoforms 2 and 4 lack residues 1–126. Compare band detection with the antibody’s binding region and the expected isoform sizes. The features alone cannot identify an observed smaller band.
UniProt describes PEG3 as a homodimer, but that annotation alone does not establish that a dimer survives Western blot preparation or causes a particular band. If a higher band appears, assess its identity experimentally before assigning it to a dimer.
Boster reagents
PEG3 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-PEG3 Antibody Picoband®
Cat # A02541-2
The catalog reports one anti-PEG3 antibody with a Western blot image. A02541-2 detected a band near 200 kDa in human HepG2 whole cell lysate; the expected size is 181 kDa. Evidence shown is limited to this example.
Which to pick: A02541-2 is the only listed option. It is catalogued for human reactivity and has a Western blot image from HepG2 lysate using 0.5 μg/mL primary antibody. Assess the approximately 200 kDa observed band against the 181 kDa expected size.
Source: BosterBio PEG3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9GZU2.
- Human Protein Atlas. PEG3 tissue expression.
- PMC5520681 — target-verified WB comparison
- PMC7099792 — target-verified WB comparison
- PMC5377817 — target-verified WB comparison