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- Table of Contents
ACSS2 / Acetyl-coenzyme A synthetase, cytoplasmic · Western blot design guide
Design a Western Blot for ACSS2
Real validated ACSS2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ACSS2 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
ACSS2 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 | ~78.6 kDa | |
| Observed band | ~79 kDa | |
| Gel | 10% (catalog A02809-1) | |
| Positive control | Adrenal gland (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 | Phosphorylated + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated ACSS2 Western Blot Protocols
The A02809-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human HepG2, human U87, human MCF-7 (catalog A02809-1) |
| Gel % | 10% (catalog A02809-1) |
| Load | 30 ug; reducing conditions (catalog A02809-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A02809-1) |
| Membrane | nitrocellulose membrane (catalog A02809-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A02809-1) |
| Primary antibody | A02809-1 · 0.25 μg/mL (catalog A02809-1) |
| Primary incubation | overnight at 4°C (catalog A02809-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A02809-1) |
| Secondary incubation | 1.5 hour at RT (catalog A02809-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A02809-1) |
| Detection | ECL (catalog A02809-1) |
Section 2
What Is the Expected ACSS2 Western Blot Band Size?
ACSS2 is predicted at 78.6 kDa and observed at ~79 kDa; the cause of the small difference is not established.
| Band at ~79 kDa | Matches the observed ACSS2 band and is close to its predicted 78.6 kDa mass. |
| Additional bands at different positions | Isoforms 1 and 2 are annotated, but distinct migration is not established. |
| Weaker cytosolic band after glucose deprivation | AMPK-dependent phosphorylation at Ser-659 can promote nuclear translocation. |
| Stronger nuclear band after glucose deprivation | ACSS2 can redistribute to the nucleus. |
💡Expected ACSS2 appearanceACSS2 is predicted at 78.6 kDa and observed at ~79 kDa; confirm band identity with an appropriate specificity control if additional bands appear.
| Predicted molecular mass | Places full-length ACSS2 near 78.6 kDa; the observed band is ~79 kDa. |
| Alternative splicing | May change protein size, but distinct migration has not been demonstrated. |
| Isoform 1 | Its individual mass and migration relative to isoform 2 are not supplied. |
| Isoform 2 | Its individual mass and migration relative to isoform 1 are not supplied. |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | The supplied features do not establish a size shift above ~79 kDa. | Check band identity with ACSS2 depletion and a second antibody. |
| Band lower than expected | Isoforms are annotated, but their masses and migration are unknown. | Check band identity with ACSS2 depletion and an isoform-specific reagent if available. |
| Multiple bands | Isoforms 1 and 2 are annotated, though separate bands are unproven. | Confirm each band with ACSS2 depletion and isoform-specific evidence. |
| Weak or no signal | ACSS2 can move from cytosol to nucleus after glucose deprivation. | Compare cytosolic and nuclear fractions with suitable fraction controls. |
| Fragments below expected size | No cleavage feature establishes an ACSS2 fragment size. | Check sample integrity and confirm any smaller band with ACSS2 depletion. |
Experiment evidence
Western blot images showing ACSS2 band
A02809-1 · ACSS2
M02809 · ACSS2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ACSS2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for ACSS2 in Western blot, you can use adrenal gland tissue, which shows medium HPA expression.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: Whole-cell lysate is suitable, but glucose deprivation may shift ACSS2 from the cytosol to the nucleus.
HPA tissue expression evidence for ACSS2
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 | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Caudate | glial cells | Medium | Protein (IHC) | HPA → |
| Colon | glandular cells | Medium | Protein (IHC) | HPA → |
| Duodenum | glandular cells | 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 → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Kidney | cells in glomeruli | Not detected | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ACSS2 Western Blot Tips
Deeper troubleshooting and optimisation questions for ACSS2, answered from its protein features.
Where should the main ACSS2 band appear?
Band shift · ACSS2 has a predicted mass of 78.6 kDa, consistent with the reported band near 79 kDa. Use that region as the initial reference; predicted mass alone does not establish band identity.
Could ACSS2 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. At UniProt position 277, isoform 2 replaces V with VQGKLKEKSKRVQP. This sequence difference could affect migration, but the feature does not establish that two bands will resolve. Check whether the antibody recognizes both isoforms.
Which phosphorylation sites matter when interpreting ACSS2 bands?
PTM · UniProt lists phosphoserines at positions 28, 30, 36, 263, 265, 267, and 659; it attributes Ser-659 phosphorylation to AMPK. Record the UniProt coordinate convention when comparing antibody or paper labels. These sites alone do not demonstrate a visible band shift.
UniProt lists N6-acetyllysine at positions 418 and 661. Note those UniProt coordinates when selecting or interpreting modification-specific antibodies. Their presence does not establish a detectable mobility shift or explain a mass difference.
UniProt lists N6-acetyllysine at positions 418 and 661. Note those UniProt coordinates when selecting or interpreting modification-specific antibodies. Their presence does not establish a detectable mobility shift or explain a mass difference.
Does this guide establish induction of ACSS2?
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 ACSS2?
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 A02809-1 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 ACSS2 be quantified across conditions?
Quantitation · Choose the same band region near 79 kDa across samples and state whether measurements come from total lysate or a cellular fraction. ACSS2 occurs in the cytosol and nucleus, and glucose deprivation can drive nuclear translocation, so fraction-specific signal changes require that context.
How might glucose deprivation affect an ACSS2 Western blot?
Interpretation · UniProt states that glucose deprivation leads to AMPK-dependent phosphorylation and subsequent nuclear translocation of ACSS2. Its Ser-659-phosphorylated form interacts with KPNA1. If comparing conditions, consider cytosolic and nuclear fractions alongside total lysate; a change in one fraction need not reflect a change in total ACSS2.
Compare them with the expected region near 79 kDa and consider the two annotated isoforms, including the sequence change at UniProt position 277. Phosphorylation and acetylation are also annotated, but their presence does not establish that they cause a visible shift. The supplied features alone cannot identify an unexpected band.
Compare them with the expected region near 79 kDa and consider the two annotated isoforms, including the sequence change at UniProt position 277. Phosphorylation and acetylation are also annotated, but their presence does not establish that they cause a visible shift. The supplied features alone cannot identify an unexpected band.
Boster reagents
ACSS2 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-ACSS2 Antibody Picoband®
Cat # A02809-1
Real WB data
Anti-ACSS2/Acetyl Coa Synthetase Rabbit Monoclonal Antibody
Cat # M02809
Both the supplier anti-ACSS2 antibodies have Western blot images showing an approximately 79 kDa band in the listed human, rat, and mouse samples. These examples document tested samples, not broader validation.
Which to pick: Both A02809-1 and M02809 list human, mouse, and rat reactivity and have WB images. Match the illustrated samples: A02809-1 includes U87, MCF-7, C6, and Neuro-2a lysates; M02809 includes K562, HEL, and kidney tissue lysates.
Source: BosterBio ACSS2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9NR19.
- Human Protein Atlas. ACSS2 tissue expression.
- PMC9883393 — target-verified WB comparison
- PMC9613512 — target-verified WB comparison
- PMC12058118 — target-verified WB comparison