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- Table of Contents
PPAT / Amidophosphoribosyltransferase · Western blot design guide
Design a Western Blot for PPAT
Real validated PPAT Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PPAT 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
PPAT 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 | ~57.4 kDa | |
| Observed band | ~57 kDa | |
| Gel | 10% (catalog A05857-2) | |
| Positive control | Soft tissue (IHC candidate; verify WB) +4 more | |
| Negative control | Bone marrow (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Acetylated + Cleaved | |
| Caveat | Maturation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated PPAT Western Blot Protocols
The A05857-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human 293T, human HepG2, human K562 (catalog A05857-2) |
| Gel % | 10% (catalog A05857-2) |
| Load | 30 ug; reducing conditions (catalog A05857-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A05857-2) |
| Membrane | nitrocellulose membrane (catalog A05857-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A05857-2) |
| Primary antibody | A05857-2 · 0.5 μg/mL (catalog A05857-2) |
| Primary incubation | overnight at 4°C (catalog A05857-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A05857-2) |
| Secondary incubation | 1.5 hour at RT (catalog A05857-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A05857-2) |
| Detection | ECL (catalog A05857-2) |
Section 2
What Is the Expected PPAT Western Blot Band Size?
PPAT is predicted at 57.4 kDa and observed near 57 kDa in reducing lysates; the cause of the small difference is not established.
| Single band near 57 kDa | matches the empirical PPAT band in reducing whole-cell lysates |
| Band near 57.4 kDa | consistent with the predicted full-length precursor mass |
| Band slightly below the precursor | could reflect removal of the annotated residues 1–11 propeptide |
| Close bands near 57 kDa | could represent precursor and processed PPAT, pending identity controls |
💡Expected PPAT appearancePPAT has a predicted mass of 57.4 kDa and an observed band near 57 kDa in reducing whole-cell lysates; confirm band identity with an independent antibody or PPAT depletion.
| UniProt predicted mass | places the 517-residue precursor near 57.4 kDa |
| Full-length precursor | provides the predicted mass before propeptide removal |
| Residues 1–11 propeptide | its removal could make mature PPAT slightly smaller than the precursor |
| Propeptide retention | could leave PPAT slightly larger than its processed form |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | PPAT is annotated as a homotetramer, which could persist if denaturation is incomplete | Repeat with thorough SDS denaturation and compare with the reducing 57 kDa reference band |
| Band lower than expected | Removal of the residues 1–11 propeptide is possible, but the band identity is unproven | Check with an independent antibody or PPAT depletion |
| Multiple bands | Precursor and processed PPAT are possible, but distinct migration is unestablished | Identify each band with PPAT depletion or an independent antibody |
| Weak or no signal | PPAT abundance or detection may be insufficient in the tested lysate | Include a positive lysate and verify loading and antibody performance |
| Fragments below expected size | Sample degradation or additional cleavage is possible but not established by the supplied features | Prepare fresh lysate with protease inhibitors and verify fragment identity |
Experiment evidence
Western blot images showing PPAT band
A05857-2 · PPAT
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PPAT Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PPAT in Western blot, you can use soft tissue, which has high HPA expression.
Positive control: Soft tissue (IHC candidate; verify WB)
Negative control: Bone marrow (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: Bone marrow is reported as not detected by HPA, but confirm its suitability as a negative control in your assay.
HPA tissue expression evidence for PPAT
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 |
|---|---|---|---|---|
| Soft tissue | chondrocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Caudate | glial cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | endothelial cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cervix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PPAT Western Blot Tips
Deeper troubleshooting and optimisation questions for PPAT, answered from its protein features.
How should PPAT 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.
Do PPAT isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It provides no isoform-based explanation for multiple bands; assess their identity independently.
Could acetylation cause a visible PPAT band shift?
PTM · UniProt lists N-acetylmethionine at position 1, using its supplied sequence coordinates. This modification alone does not establish a visible shift or explain a difference from the calculated mass.
Does this guide establish induction of PPAT?
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 PPAT?
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 A05857-2 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 I quantify the PPAT band?
Quantitation · Use the band near the reported 57 kDa position for consistent comparisons across samples. If additional bands appear, establish their identity before including them in PPAT quantitation.
Why is the observed PPAT band near its predicted mass?
Interpretation · PPAT’s predicted mass is 57.4 kDa, and the reported band is about 57 kDa. That agreement supports identifying the band as PPAT, but mass alone does not establish identity.
UniProt annotates residues 1–11 as a propeptide. Processing could affect apparent size, but the annotation alone does not show that processing occurred in your sample or that it caused a visible shift.
PPAT is annotated as a homotetramer. Check whether a higher band persists when samples are prepared to dissociate protein complexes, but the oligomer annotation alone does not identify that band.
Consider the annotated propeptide at residues 1–11 when assessing processing, and verify the lower band’s identity. The feature does not establish that cleavage occurred or that it produced the observed band.
UniProt annotates residues 1–11 as a propeptide. Processing could affect apparent size, but the annotation alone does not show that processing occurred in your sample or that it caused a visible shift.
PPAT is annotated as a homotetramer. Check whether a higher band persists when samples are prepared to dissociate protein complexes, but the oligomer annotation alone does not identify that band.
Consider the annotated propeptide at residues 1–11 when assessing processing, and verify the lower band’s identity. The feature does not establish that cleavage occurred or that it produced the observed band.
Boster reagents
PPAT 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-PPAT Antibody Picoband®
Cat # A05857-2
The catalog reports one anti-PPAT antibody, A05857-2. Its supplied Western blot shows a band near the expected 57 kDa in human 293T, HepG2, and K562, rat PC-12, and mouse RAW264.7 whole-cell lysates. The supplied evidence is limited to this product image.
Which to pick: A05857-2 is the only listed option. It is catalogued for human, mouse, and rat and has a Western blot image using the named cell lysates at 30 µg per lane under reducing conditions.
Source: BosterBio PPAT gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q06203.
- Human Protein Atlas. PPAT tissue expression.
- PMC11531959 — target-verified WB comparison
- PMC7145547 — target-verified WB comparison
- PMC4695129 — target-verified WB comparison