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- Table of Contents
ACAT1 / Acetyl-CoA acetyltransferase, mitochondrial · Western blot design guide
Design a Western Blot for ACAT1
Real validated ACAT1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ACAT1 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
ACAT1 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 | ~45.2 kDa | |
| Observed band | ~40 kDa | |
| Gel | 5–20% (catalog A02008-1) | |
| 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 | Acetylated | |
| Caveat | Band identity controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated ACAT1 Western Blot Protocols
The A02008-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human HepG2, human PC-3, human A549 (catalog A02008-1) |
| Gel % | 5–20% (catalog A02008-1) |
| Load | 30 ug; reducing conditions (catalog A02008-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A02008-1) |
| Membrane | nitrocellulose membrane (catalog A02008-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A02008-1) |
| Primary antibody | A02008-1 · 0.5 μg/mL (catalog A02008-1) |
| Primary incubation | overnight at 4°C (catalog A02008-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A02008-1) |
| Secondary incubation | 1.5 hour at RT (catalog A02008-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A02008-1) |
| Detection | ECL (catalog A02008-1) |
Section 2
What Is the Expected ACAT1 Western Blot Band Size?
ACAT1 is predicted at 45.2 kDa and observed near 40 kDa; the cause of this difference is not established by the supplied evidence.
| Band near 40 kDa | Empirical ACAT1 band; confirm identity with band controls |
| Band near 45.2 kDa | Near the predicted full-length mass, without demonstrated migration at this position |
| Several bands at different sizes | Isoforms 1 and 2 are annotated, but distinct bands are not established |
| Weak or absent band in a mitochondria-poor sample | ACAT1 is mitochondrial |
💡Expected ACAT1 appearanceACAT1 has a predicted mass of 45.2 kDa and an empirical band near 40 kDa; the cause of the difference is unestablished, so confirm identity with ordinary band controls.
| Predicted full-length mass | 45.2 kDa is the sequence-based reference; the empirical band is near 40 kDa |
| Mitochondrial transit peptide | Processing could lower the mass, but its extent and contribution to the 40 kDa band are unestablished |
| 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 | Low mitochondrial content in the sample | Check mitochondrial enrichment and use a positive lysate |
| Band higher than expected | A precursor or another protein may be detected | Compare with the 40 kDa reference and verify antibody specificity |
| Band lower than expected | The documented ACAT1 band is near 40 kDa, below its predicted 45.2 kDa mass | Compare with a positive lysate and verify band identity |
| Broad smear instead of sharp band | Sample quality or nonspecific signal; no glycosylation sites are listed | Check sample integrity and antibody specificity |
| Multiple bands | Isoforms are annotated, but their migration is unestablished | Check band identity before assigning either band to an isoform |
| Fragments below expected size | Possible sample degradation | Check sample integrity and compare with a fresh positive lysate |
Experiment evidence
Western blot images showing ACAT1 band
A02008-1 · ACAT1
M02008 · ACAT1
M02008 · ACAT1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ACAT1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for ACAT1 in Western blot, you can use adrenal gland tissue.
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 the samples.
⚠️Feasibility: No HPA tissue is listed as not detected, so use siRNA knockdown or a KO line for a negative control.
HPA tissue expression evidence for ACAT1
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 → |
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Smooth muscle | smooth muscle cells | Low | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Breast | glandular cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | endothelial cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced ACAT1 Western Blot Tips
Deeper troubleshooting and optimisation questions for ACAT1, answered from its protein features.
How should ACAT1 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 ACAT1 isoforms produce different Western blot bands?
Isoforms · Yes. Isoform 2 replaces canonical residues 146–162 and lacks residues 163–427, so its sequence differs substantially from isoform 1. The supplied features do not establish its apparent Western blot mass or whether it is expressed in your sample.
Choose an epitope within canonical residues 1–145, which precede isoform 2’s sequence replacement and deletion. An epitope in residues 163–427 would miss isoform 2. Check the antibody’s stated epitope against the sequences.
Choose an epitope within canonical residues 1–145, which precede isoform 2’s sequence replacement and deletion. An epitope in residues 163–427 would miss isoform 2. Check the antibody’s stated epitope against the sequences.
Which ACAT1 modifications matter when interpreting bands?
PTM · UniProt lists acetyllysine at 66, 174, 181, 190, 202, 223, 230, 251, 257, 263, 273, and 338; succinyllysine at 66, 78, 174, 181, 190, 202, 223, 230, 243, 263, 266, and 268. These are canonical UniProt coordinates; antibody or paper numbering may differ. Several sites have alternate modifications. Their presence does not establish a visible shift.
No glycosylation sites are supplied for ACAT1, so these features do not support a glycosylation-based explanation. Compare the apparent band with the supplied approximately 40 kDa observation and confirm its identity experimentally.
No glycosylation sites are supplied for ACAT1, so these features do not support a glycosylation-based explanation. Compare the apparent band with the supplied approximately 40 kDa observation and confirm its identity experimentally.
Does this guide establish induction of ACAT1?
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 ACAT1?
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 A02008-1 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 ACAT1 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.
Why might ACAT1 appear near 40 kDa instead of 45.2 kDa?
Interpretation · The supplied apparent band is about 40 kDa, while the predicted mass is 45.2 kDa. ACAT1 has a transit peptide keyword, but no cleavage coordinates are supplied. The features alone cannot establish why the masses differ; compare the band with an ACAT1-specific control.
ACAT1 is mitochondrial. Compare samples prepared with the same fractionation method and normalize consistently; differences in mitochondrial recovery could affect the measured signal. Quantify the same validated band across samples.
First check whether the antibody epitope is present in each isoform. Isoform 2 has a replacement at 146–162 and lacks 163–427. ACAT1 is also reported as a homotetramer, but that feature alone does not identify a higher band. The listed modifications likewise do not establish a visible shift. Confirm band identity with an ACAT1-specific control.
ACAT1 is mitochondrial. Compare samples prepared with the same fractionation method and normalize consistently; differences in mitochondrial recovery could affect the measured signal. Quantify the same validated band across samples.
First check whether the antibody epitope is present in each isoform. Isoform 2 has a replacement at 146–162 and lacks 163–427. ACAT1 is also reported as a homotetramer, but that feature alone does not identify a higher band. The listed modifications likewise do not establish a visible shift. Confirm band identity with an ACAT1-specific control.
Boster reagents
ACAT1 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-ACAT1 Antibody Picoband®
Cat # A02008-1
Real WB data
Anti-ACAT1 Rabbit Monoclonal Antibody
Cat # M02008
Two anti-ACAT1 antibodies list human, mouse, and rat reactivity. A02008-1 has a Western blot caption showing an approximately 40 kDa band in specified human cell, rat tissue, and mouse heart lysates. M02008’s image caption describes immunoprecipitation with a WB step.
Which to pick: Choose A02008-1 if you want a documented standalone Western blot example for the listed specimens and conditions. M02008 is also listed for human, mouse, and rat, but its supplied image evidence describes immunoprecipitation with WB at 1:1,000.
Source: BosterBio ACAT1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P24752.
- Human Protein Atlas. ACAT1 tissue expression.
- PMC11544177 — target-verified WB comparison
- PMC13470199 — target-verified WB comparison
- PMC11377227 — target-verified WB comparison