This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
DBT / Lipoamide acyltransferase component of branched-chain alpha-keto acid dehydrogenase complex, mitochondrial · Western blot design guide
Design a Western Blot for DBT
Real validated DBT Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-DBT 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
DBT 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 | ~53.5 kDa | |
| Observed band | ~53 kDa | |
| Gel | 5–20% (catalog A01145-2) | |
| Positive control | Adrenal gland (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 | Phosphorylated + Acetylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated DBT Western Blot Protocols
The A01145-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human SiHa, human A431, human Hacat (catalog A01145-2) |
| Gel % | 5–20% (catalog A01145-2) |
| Load | 30 ug; reducing conditions (catalog A01145-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A01145-2) |
| Membrane | nitrocellulose membrane (catalog A01145-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A01145-2) |
| Primary antibody | A01145-2 · 0.5 μg/mL (catalog A01145-2) |
| Primary incubation | overnight at 4°C (catalog A01145-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A01145-2) |
| Secondary incubation | 1.5 hour at RT (catalog A01145-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A01145-2) |
| Detection | ECL (catalog A01145-2) |
Section 2
What Is the Expected DBT Western Blot Band Size?
DBT is predicted at 53.5 kDa and observed at ~53 kDa; the cause of the small difference is not established.
| Band at ~53 kDa | empirical DBT band, close to its 53.5 kDa predicted mass |
| Single sharp band near 53 kDa | consistent with DBT; confirm identity with antibody controls |
| High-mass retained signal | could reflect incomplete dissociation of the DBT structural core |
| Weak or absent band in lysate | could reflect low recovery of mitochondrial matrix DBT |
💡Expected DBT appearanceDBT has a predicted mass of 53.5 kDa and an empirical band at ~53 kDa; confirm band identity with appropriate antibody controls, since the supplied features do not establish the cause of the small difference.
| Predicted DBT mass | 53.5 kDa provides the sequence-based reference |
| N6-lipoyllysine at residue 105 | adds a modification without an established visible migration shift |
| N6-succinyllysine at residue 133 | may affect mobility, but no shift is established |
| Alternate acetylation or succinylation at residue 196 | creates modified states without a demonstrated band separation |
| Phosphoserine at residue 220 | may affect mobility, but no shift is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | poor recovery of mitochondrial matrix DBT | check mitochondrial enrichment and sample loading |
| Band higher than expected | possible incomplete dissociation of the DBT structural core | check denaturation conditions and compare with a fully denatured sample |
| Band lower than expected | band identity or protein integrity is uncertain | check with a second DBT antibody and inspect sample handling |
| Multiple bands | additional bands are not established by the supplied DBT features | compare with a DBT depletion control or a second antibody |
| Weak or no signal | low mitochondrial DBT recovery or insufficient detection | check loading, transfer, and mitochondrial enrichment |
| Fragments below expected size | possible sample degradation, not an established DBT processing product | prepare fresh lysate with protease inhibitors and confirm with a second epitope |
Experiment evidence
Western blot images showing DBT band
A01145-2 · DBT
A01145-2 · DBT
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for DBT Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for DBT in Western blot, you can use adrenal gland lysate, which has high HPA expression.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Bone marrow (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside DBT.
⚠️Feasibility: DBT is mitochondrial, so its signal may be diluted in whole-tissue lysate.
HPA tissue expression evidence for DBT
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 → |
| Kidney | collecting ducts | High | Protein (IHC) | HPA → |
| Pancreas | exocrine glandular cells | High | Protein (IHC) | HPA → |
| Stomach | glandular cells | High | Protein (IHC) | HPA → |
| Appendix | glandular 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 → |
| Ovary | follicle cells | Not detected | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Not detected | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Low | Protein (IHC) | HPA → |
| Bronchus | ciliated cells (cell body) | Low | Protein (IHC) | HPA → |
Section 3
Advanced DBT Western Blot Tips
Deeper troubleshooting and optimisation questions for DBT, answered from its protein features.
How should DBT 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 a second DBT band be an annotated isoform?
Isoforms · Only one isoform is supplied, with no alternative sequence. Do not assign an additional band to a DBT isoform on this evidence alone.
Which DBT modifications could vary between samples?
PTM · UniProt lists alternate acetylation or succinylation at Lys196, Lys289, and Lys440, as well as other modified residues. These annotations identify possible modification states; they do not predict separate visible bands.
Yes. UniProt lists phosphoserine at Ser220, using its sequence numbering. A change in a phospho-specific signal would need to be distinguished from a change in total DBT; this feature alone does not predict a band shift.
Yes. UniProt lists phosphoserine at Ser220, using its sequence numbering. A change in a phospho-specific signal would need to be distinguished from a change in total DBT; this feature alone does not predict a band shift.
Does this guide establish induction of DBT?
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 DBT?
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 A01145-2 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 DBT be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Does the observed DBT band match its predicted mass?
Interpretation · The reported band at approximately 53 kDa is close to the predicted 53.5 kDa. The listed modifications do not, by themselves, establish a visible shift or explain any small difference in apparent mass.
UniProt lists N6-lipoyllysine at Lys105. Use UniProt sequence numbering when comparing this site with antibody or paper numbering, which may differ.
Measure the same approximately 53 kDa band under consistent sample preparation and normalize for loading. DBT is annotated in the mitochondrial matrix, so comparisons between whole-cell and mitochondrial preparations also need to account for their different sample composition.
DBT forms a 24-polypeptide structural core, so incomplete dissociation is one possibility to investigate. The oligomer annotation alone cannot identify an unexpected band; compare sample preparation conditions and confirm band identity before assigning it to DBT.
UniProt lists N6-lipoyllysine at Lys105. Use UniProt sequence numbering when comparing this site with antibody or paper numbering, which may differ.
Measure the same approximately 53 kDa band under consistent sample preparation and normalize for loading. DBT is annotated in the mitochondrial matrix, so comparisons between whole-cell and mitochondrial preparations also need to account for their different sample composition.
DBT forms a 24-polypeptide structural core, so incomplete dissociation is one possibility to investigate. The oligomer annotation alone cannot identify an unexpected band; compare sample preparation conditions and confirm band identity before assigning it to DBT.
Boster reagents
DBT 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-DBT Antibody Picoband®
Cat # A01145-2
The catalog reports one anti-DBT antibody, A01145-2. Its Western blot caption reports an approximately 53 kDa band in human cell lysates and rat and mouse tissue lysates. No independent validation or publication evidence is supplied.
Which to pick: A01145-2 is the only listed option. Its WB image includes human SiHa, A431, Hacat and Jurkat cells, plus rat and mouse stomach and kidney tissues. Choose it when those reported sample contexts are relevant to your experiment.
Source: BosterBio DBT gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P11182.
- Human Protein Atlas. DBT tissue expression.
- PMC9666696 — target-verified WB comparison