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KAT2A · Western blot design guide
Design a Western Blot for KAT2A
Real validated KAT2A Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-KAT2A WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
KAT2A 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 | ~93.9 kDa | |
| Observed band | ~94 kDa | |
| Gel | 8–10% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Homooligomer formation | |
| Regulation | Notch signaling | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated KAT2A Western Blot Protocols
Literature-validated Western blot parameters for KAT2A — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human CACO-2 , Lane 2: rat C6 , Lane 3: mouse brain . After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-KAT2A/GCN5 antigen affinity purified polyclonal antibody (PB9713) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for KAT2A/GCN5 at approximately 94 kDa. The expected band size for KAT2A/GCN5 is at 94 kDa |
| Gel % | 8–10% |
| Load | 30 ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 94 kDa |
Section 2
What Is the Expected KAT2A Western Blot Band Size?
KAT2A has a 93.9 kDa predicted mass and is observed at approximately 94 kDa, a close match reflecting the absence of glycosylation, cleavage, or disulfide-linked oligomerization.
| Single band at approximately 94 kDa | matches the 93.9 kDa predicted mass for KAT2A almost exactly, since there is no glycosylation, cleavage, or covalent crosslinking to shift the monomer |
| Faint second band at a slightly different mass | may reflect detection of splice isoform 2 alongside the canonical isoform 1 |
| Band position unchanged between reducing and non-reducing sample buffer | consistent with no annotated inter-chain disulfide bonds, so the homodimer/tetramer-of-homodimers assembly reported for KAT2A does not survive SDS denaturation and runs as monomer |
| No shift after PNGase F or O-glycosidase treatment | KAT2A has no annotated N- or O-glycosylation sites, so deglycosylation should not change the band position |
| Signal concentrated in nuclear/chromatin fraction, weak in cytoplasmic fraction | KAT2A is predominantly nuclear and chromosome-associated, with only a minor cytoskeletal/centrosomal pool |
💡Expected KAT2A appearanceKAT2A is expected as a single sharp band at approximately 94 kDa, closely matching its 93.9 kDa predicted mass with no glycosylation or proteolytic cleavage to shift it.
| Predicted mass (93.9 kDa) | sets the baseline expectation and closely matches the empirically observed ~94 kDa band |
| Splice isoforms 1 and 2 | isoform 2 may appear as an additional band distinct from canonical isoform 1, though no isoform-specific mass is defined |
| Homooligomerization (dimer/tetramer of homodimers) | KAT2A assembles into higher-order complexes in cells, but with no annotated inter-chain disulfide bonds this association is non-covalent and dissociates under standard SDS-PAGE, so the monomer band is expected rather than a dimer- or tetramer-sized band |
| Absence of glycosylation sites | favors a sharp, tight band rather than a smear, since there are no N- or O-linked glycans adding heterogeneous mass |
| No signal peptide or propeptide | no precursor cleavage occurs, so only the full-length ~94 kDa form is expected, not a separate precursor/mature pair |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | incomplete denaturation leaving residual homooligomer association, or cross-reactivity with related bromodomain/HAT family proteins | boil samples longer in fresh reducing sample buffer and confirm specificity with a KAT2A knockdown or knockout control |
| Band lower than expected | partial proteolytic degradation of this large 837-residue multidomain protein during lysis | use fresh lysate with protease inhibitors, keep samples cold, and avoid repeated freeze-thaw cycles |
| Multiple bands | detection of both splice isoforms or cross-reactivity with related GCN5/PCAF-family acetyltransferases | compare band pattern to isoform-specific antibody data or a knockout control to confirm which bands are specific |
| Weak or no signal | KAT2A is predominantly nuclear and chromatin-associated, so standard whole-cell lysis can under-extract the chromatin-bound pool | use a nuclear or chromatin extraction protocol and load a nuclear-enriched fraction |
| Broad smear instead of sharp band | since KAT2A has no annotated glycosylation, a smear more likely indicates degradation, aggregation, or overloading than glycoform heterogeneity | reduce protein loading, add fresh protease inhibitors, and avoid excessive heating time |
| Fragments below expected size | the large multidomain HAT/bromodomain structure of KAT2A is prone to proteolytic clipping during extraction | minimize handling time, keep lysates cold, and include a protease inhibitor cocktail throughout preparation |
Experiment evidence
Western blot images showing KAT2A band
PB9713 · KAT2A
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for KAT2A Western blot
🧪For positive controls for KAT2A in Western blot, you can use HeLa cell lysate, since KAT2A is a broadly expressed nuclear histone acetyltransferase found in standard human cell lines.
Positive control: HeLa cell lysate
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin as loading controls alongside a total-protein stain such as stain-free gel imaging, Ponceau S, or REVERT.
⚠️Feasibility: As a ubiquitously expressed nuclear protein with no HPA tissue data available to identify a clean negative, specificity is best confirmed using an siRNA knockdown or KO cell line rather than a tissue-based negative control.
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced KAT2A Western Blot Tips
Deeper troubleshooting and optimisation questions for KAT2A, answered from its protein features.
Why does the observed KAT2A band match predicted mass?
KAT2A has no signal peptide, propeptide, or glycosylation to alter mobility, so its predicted mass of 93.9 kDa closely matches the observed ~94 kDa band under standard denaturing SDS-PAGE conditions.
Could isoform 2 produce a different molecular weight?
UniProt lists two KAT2A isoforms (1 and 2) from alternative splicing. Isoform 2 may lack exons present in isoform 1, producing a lower-molecular-weight band alongside or instead of the ~94 kDa canonical band, depending on tissue and antibody epitope coverage.
Do phosphorylation or acetylation shift the KAT2A band?
KAT2A carries four modified residues, including phosphorylation and acetylation. These modifications add minimal mass and typically do not cause a visible shift on standard SDS-PAGE, though a hyperphosphorylated pool may appear as a slightly diffuse or doublet band near 94 kDa.
Does ubiquitin-like conjugation affect KAT2A protein levels?
KAT2A undergoes Ubl conjugation via isopeptide bond formation, consistent with SUMOylation or ubiquitination that can regulate its stability and turnover. Cells with altered SUMO/ubiquitin pathway activity may show reduced or absent KAT2A signal from proteasomal degradation rather than loss of expression.
What transfer method to use for KAT2A Western blot?
At ~94 kDa, use wet transfer for improved efficiency of high-molecular-weight proteins, or extend semi-dry transfer time. Use 0.45 µm PVDF or nitrocellulose to prevent blow-through, with 10-20% methanol in wet transfer buffer and adequate transfer time of 60-90 minutes.
What blocking approach suits KAT2A detection?
KAT2A has no glycosylation, so lectin-based or glyco-specific blocking concerns do not apply. Standard 5% non-fat milk or BSA in TBST is sufficient; use BSA if downstream phospho-specific antibody probing is planned, since milk contains phosphoproteins that can raise background.
How should KAT2A be quantified relative to loading controls?
Since KAT2A localizes mainly to the nucleus and chromosome, normalize using a nuclear loading control such as Lamin B1 or Histone H3 rather than a cytoplasmic control like GAPDH, especially when comparing nuclear-enriched fractions or chromatin extracts.
What explains extra bands above the 94 kDa band?
KAT2A forms homooligomers, potentially tetramers of homodimers, and complexes with EP300, CREBBP, ADA2, and TFTC-HAT subunits. Incomplete denaturation or insufficient reducing conditions can leave residual oligomers or complex components, producing higher-molecular-weight bands beyond the ~94 kDa monomer.
Boster reagents
Best KAT2A Western Blot Antibodies
BosterBio's KAT2A antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-KAT2A/GCN5 Antibody Picoband®
Cat # PB9713
These anti-KAT2A/GCN5 antibodies are our best-performing, most widely cited reagents, thoroughly validated by western blot and cross-validated against negative-tissue controls and complementary methods to confirm specific, reproducible detection of endogenous KAT2A/GCN5.
Which to pick: Only one Boster KAT2A/GCN5 antibody is listed, PB9713, which includes a validated western blot image (10% SDS-PAGE) confirming specific KAT2A/GCN5 detection—making it the clear, single recommended choice for your western blot.
Source: BosterBio KAT2A gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q92830.
- Human Protein Atlas. KAT2A tissue expression.