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PNPLA2 / Patatin-like phospholipase domain-containing protein 2 · Western blot design guide
Design a Western Blot for PNPLA2
Real validated PNPLA2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PNPLA2 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
PNPLA2 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 | ~55.3 kDa | |
| Observed band | ~55 kDa | |
| Gel | 5–20% (catalog M01800) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Oral mucosa (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated PNPLA2 Western Blot Protocols
The M01800 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human A431, human 293T, human SiHa (catalog M01800) |
| Gel % | 5–20% (catalog M01800) |
| Load | 30 ug; reducing conditions (catalog M01800) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M01800) |
| Membrane | nitrocellulose membrane (catalog M01800) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M01800) |
| Primary antibody | M01800 · 1:500 (catalog M01800) |
| Primary incubation | overnight at 4°C (catalog M01800) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:1000 (catalog M01800) |
| Secondary incubation | 1.5 hour at RT (catalog M01800) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M01800) |
| Detection | ECL (catalog M01800) |
Section 2
What Is the Expected PNPLA2 Western Blot Band Size?
PNPLA2 is predicted at 55.3 kDa and observed at ~55 kDa; their small difference has no established cause in the supplied evidence.
| Band at ~55 kDa | Matches the empirical PNPLA2 band and its 55.3 kDa predicted mass |
| Band above ~55 kDa | Could reflect Asn39 N-linked glycosylation, but a visible shift is unproven |
| Multiple bands near ~55 kDa | Isoforms 1 and 2 are possible contributors; their migration is unknown |
| Weak band in whole-cell lysate | PNPLA2 localizes to lipid droplets and membranes, which may affect recovery |
💡Expected PNPLA2 appearancePNPLA2 has a predicted mass of 55.3 kDa and an empirical band at ~55 kDa; confirm band identity with appropriate positive and knockdown controls.
| UniProt predicted mass | The 55.3 kDa sequence mass agrees with the empirical ~55 kDa band |
| Asn39 N-linked glycosylation site | May affect apparent size if occupied; no visible shift is established |
| Splice isoform 1 | May migrate differently from isoform 2; its individual size is unknown |
| Splice isoform 2 | May migrate differently from isoform 1; its individual size is unknown |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Lipid-droplet or membrane-associated PNPLA2 may be poorly recovered | Check extraction and an enriched fraction with a positive control |
| Band higher than expected | Asn39 glycosylation is possible, but its migration effect is unproven | Compare with deglycosylated sample and a knockdown control |
| Band lower than expected | The identity of a lower band is unestablished; isoform sizes are unknown | Check antibody specificity with PNPLA2 knockdown and isoform controls |
| Multiple bands | Isoforms 1 and 2 exist, but distinct bands are unconfirmed | Compare isoform expression and PNPLA2 knockdown samples |
| Weak or no signal | Recovery of lipid-droplet or membrane-associated PNPLA2 may be low | Check loading, extraction, and a positive-control lysate |
Experiment evidence
Western blot images showing PNPLA2 band
M01800 · PNPLA2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PNPLA2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PNPLA2 in Western blot, you can use adrenal gland tissue, which has high HPA expression.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Oral mucosa (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: HPA reports high expression in adrenal gland and no detection in oral mucosa, making tissue controls feasible.
HPA tissue expression evidence for PNPLA2
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 → |
| Duodenum | glandular cells | High | Protein (IHC) | HPA → |
| Nasopharynx | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Pancreas | exocrine glandular cells | High | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Oral mucosa | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Breast | glandular cells | Low | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Low | Protein (IHC) | HPA → |
| Cervix | squamous epithelial cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced PNPLA2 Western Blot Tips
Deeper troubleshooting and optimisation questions for PNPLA2, answered from its protein features.
How should PNPLA2 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 PNPLA2 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. Isoform 2 lacks canonical residues 1–324, so it may produce a smaller band if expressed and recognized by the antibody. Check whether the antibody epitope lies within that missing region before interpreting a second band.
Which PNPLA2 phosphorylation sites matter for band interpretation?
PTM · UniProt lists phosphoserine at canonical Ser372 (in vitro), Ser404 (by PKA and FAM20C), and Ser428. These sites may affect antibody recognition, especially for a phospho-specific antibody, but their presence does not establish a visible mobility shift. Check the antibody’s numbering convention before comparing sites.
UniProt lists one N-linked glycosylation site at canonical Asn39. That residue is absent from isoform 2. The site annotation alone does not show that a sample is glycosylated or explain a mass difference; compare treated and untreated samples if testing its effect.
UniProt lists one N-linked glycosylation site at canonical Asn39. That residue is absent from isoform 2. The site annotation alone does not show that a sample is glycosylated or explain a mass difference; compare treated and untreated samples if testing its effect.
Could PKA stimulation change PNPLA2 Western blot results?
Induction · Canonical Ser404 is annotated as phosphorylated by PKA and FAM20C. A PKA-related condition could therefore be relevant when assessing Ser404 phosphorylation, but the annotation does not establish a change in total PNPLA2 abundance or band mobility. Compare matched conditions with appropriate loading controls.
What transfer method to use for PNPLA2 Western blot?
Transfer · PNPLA2 is annotated at lipid droplets and the cell membrane, with a predicted transmembrane region. Check extraction and transfer recovery by examining the post-transfer gel and membrane. These features do not establish whether wet or semi-dry transfer works better for a given sample.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M01800 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 PNPLA2 bands be quantified?
Quantitation · Quantify the approximately 55 kDa band with consistent sample preparation and loading normalization. PNPLA2 is found at lipid droplets, the cell membrane, and in the cytoplasm, so keep the fraction analyzed consistent across samples. Its interaction with ABHD5 stimulates triglyceride hydrolase activity; band intensity alone does not measure that activity.
Does PNPLA2 migrate at its predicted mass?
Interpretation · The observed band is approximately 55 kDa, close to the predicted 55.3 kDa. A difference of this size does not require a modification-based explanation; the listed features alone do not establish a visible shift.
First compare them with the approximately 55 kDa band and check antibody specificity. Isoform 2 lacks canonical residues 1–324, including Asn39, and could be recognized differently depending on the epitope. The listed glycosylation and phosphorylation sites do not, by themselves, identify an unexpected band.
First compare them with the approximately 55 kDa band and check antibody specificity. Isoform 2 lacks canonical residues 1–324, including Asn39, and could be recognized differently depending on the epitope. The listed glycosylation and phosphorylation sites do not, by themselves, identify an unexpected band.
Boster reagents
PNPLA2 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-ATGL / PNPLA2 Rabbit Monoclonal Antibody
Cat # M01800
The catalog reports one anti-PNPLA2 antibody, M01800, with reported human, mouse, and rat reactivity. Its WB image shows an approximately 55 kDa band in the named cell and heart lysates. The supplied evidence includes no independent publication validation.
Which to pick: M01800 is the only listed option. Its WB image includes human A431, 293T, SiHa, and HepG2 cells; rat C6 cells and heart; and mouse NIH/3T3 cells and heart. Use those tested contexts to judge fit for your sample.
Source: BosterBio PNPLA2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q96AD5.
- Human Protein Atlas. PNPLA2 tissue expression.
- PMC8386627 — target-verified WB comparison
- PMC10406976 — target-verified WB comparison
- PMC12147166 — target-verified WB comparison