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PDK3 / [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 3, mitochondrial · Western blot design guide
Design a Western Blot for PDK3
Source-linked PDK3 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PDK3 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
PDK3 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 | ~46.9 kDa | |
| Observed band | ~47 kDa | |
| Gel | 5–20% (catalog A05658-1) | |
| Positive control | Cerebral cortex (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | — | |
| Caveat | — | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked PDK3 Western Blot Protocol Options
The A05658-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human MCF-7, human 293T, human RT4, human SiHa, human Jurkat, human U251, human A549, rat testis, mouse testis (catalog A05658-1) |
| Gel % | 5–20% (catalog A05658-1) |
| Load | 30 ug; reducing conditions (catalog A05658-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A05658-1) |
| Membrane | nitrocellulose membrane (catalog A05658-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A05658-1) |
| Primary antibody | A05658-1 · 0.5 μg/mL (catalog A05658-1) |
| Primary incubation | overnight at 4°C (catalog A05658-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A05658-1) |
| Secondary incubation | 1.5 hour at RT (catalog A05658-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A05658-1) |
| Detection | ECL (catalog A05658-1) |
Section 2
What Is the Expected PDK3 Western Blot Band Size?
PDK3 is predicted at 46.9 kDa and observed at approximately 47 kDa; no distinct cause for this small difference is established.
| Band at approximately 47 kDa | Observed PDK3 band, close to the 46.9 kDa predicted mass; confirm identity with a PDK3 depletion control |
| Additional bands at different positions | Isoforms 1 and 2 could differ in size, but their migration is not established |
| Higher band under incompletely denaturing conditions | The PDK3 homodimer could persist; no dimer band is demonstrated |
| Weak band in a mitochondria-depleted sample | PDK3 is located in the mitochondrial matrix |
💡Expected PDK3 appearancePDK3 has a predicted mass of 46.9 kDa and an observed band at approximately 47 kDa; confirm band identity with antibody specificity controls.
| Predicted PDK3 mass | 46.9 kDa, consistent with the observed band at approximately 47 kDa |
| Homodimer formation | Could produce a higher band if the complex survives sample preparation; none is demonstrated |
| Isoform 1 | Its size relative to isoform 2 is not supplied |
| Isoform 2 | Its size relative to isoform 1 is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Poor recovery of mitochondrial PDK3 | Check mitochondrial recovery and test an enriched fraction |
| Band higher than expected | Possible persistence of the PDK3 homodimer | Compare thoroughly denatured samples and verify the band with PDK3 depletion |
| Band lower than expected | Band identity is uncertain; isoform masses are not supplied | Check whether the band decreases after PDK3 depletion |
| Multiple bands | Isoforms 1 and 2 are annotated, but their migration is unknown | Use PDK3 depletion to identify specific bands |
| Weak or no signal | The lysate may contain little mitochondrial PDK3 | Check mitochondrial recovery and increase the amount of suitable sample |
Experiment evidence
Western blot images showing PDK3 band
A05658-1 · PDK3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PDK3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PDK3 in Western blot, you can use cerebral cortex tissue, which has medium HPA expression.
Positive control: Cerebral cortex (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: PDK3 is in the mitochondrial matrix, so mitochondrial enrichment may improve detection.
HPA tissue expression evidence for PDK3
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 |
|---|---|---|---|---|
| Cerebral cortex | neuronal cells | Medium | Protein (IHC) | HPA → |
| Duodenum | glandular cells | Medium | Protein (IHC) | HPA → |
| Endometrium | glandular cells | Medium | Protein (IHC) | HPA → |
| Hippocampus | neuronal cells | Medium | Protein (IHC) | HPA → |
| Liver | hepatocytes | 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 → |
| Bronchus | basal cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Colon | endothelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PDK3 Western Blot Tips
Deeper troubleshooting and optimisation questions for PDK3, answered from its protein features.
Does mitochondrial targeting affect the expected PDK3 band?
Band shift · PDK3 is located in the mitochondrial matrix and carries a transit peptide keyword. No cleavage coordinate or mature protein mass is supplied, so do not assign a lower band to transit peptide removal from these features alone.
Could PDK3 isoforms produce different bands?
Isoforms · PDK3 has two listed isoforms. In isoform 2, residue Q406 is replaced by QDKIKTNRTF, extending the sequence by nine residues. This could change its calculated mass, but the features do not establish whether the isoforms resolve as separate bands.
Do annotated modifications prove a band shift?
PTM · The linked UniProt record describes protein features. A modification annotation alone does not demonstrate a visible shift; retain any condition or experimental qualifier attached to it.
Does this guide establish induction of PDK3?
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 PDK3?
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 A05658-1 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 PDK3 be quantified across samples?
Quantitation · Quantify the band near 47 kDa using consistent sample preparation and loading normalization. Because PDK3 is a mitochondrial matrix protein associated with the pyruvate dehydrogenase complex, keep mitochondrial recovery and extraction conditions comparable across samples.
Why does PDK3 run near 47 kDa?
Interpretation · The observed band near 47 kDa is close to PDK3’s predicted 46.9 kDa mass. The supplied features do not establish a cause for any small difference between apparent and calculated mass.
UniProt lists N6-succinyllysine at position 278, using its supplied sequence numbering. The site establishes a modification, but does not show that it causes a visible Western blot shift or explain a particular band.
PDK3 is listed as a homodimer and as part of the multimeric pyruvate dehydrogenase complex. Those associations alone do not identify a high band on a Western blot. Check whether the band is PDK3 before assigning it to a dimer or complex.
PDK3 is located in the mitochondrial matrix. A mitochondrial fraction can concentrate the relevant compartment; compare fractions with consistent preparation when interpreting PDK3 abundance.
UniProt lists N6-succinyllysine at position 278, using its supplied sequence numbering. The site establishes a modification, but does not show that it causes a visible Western blot shift or explain a particular band.
PDK3 is listed as a homodimer and as part of the multimeric pyruvate dehydrogenase complex. Those associations alone do not identify a high band on a Western blot. Check whether the band is PDK3 before assigning it to a dimer or complex.
PDK3 is located in the mitochondrial matrix. A mitochondrial fraction can concentrate the relevant compartment; compare fractions with consistent preparation when interpreting PDK3 abundance.
Boster reagents
PDK3 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-PDK3 Antibody Picoband®
Cat # A05658-1
The catalog reports one anti-PDK3 antibody for Western blot, A05658-1. Its product blot caption reports a band at approximately 47 kDa using human cell lysates and rat and mouse testis lysates. Independent specificity validation is not supplied.
Which to pick: A05658-1 is the only listed PDK3 antibody. It is catalogued for human, mouse, and rat and has a WB image with human cell lines and rat and mouse testis. Those tested samples do not establish performance in every tissue.
Source: BosterBio PDK3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q15120.
- Human Protein Atlas. PDK3 tissue expression.
- PMC8692537 — target-verified WB comparison
- PMC6294108 — target-verified WB comparison
- PMC6732365 — target-verified WB comparison