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- Table of Contents
SIX2 / Homeobox protein SIX2 · Western blot design guide
Design a Western Blot for SIX2
Real validated SIX2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SIX2 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
SIX2 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 | ~32.3 kDa | |
| Gel | 12–15% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | — | |
| Caveat | Peptide-blocking control | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated SIX2 Western Blot Protocols
The A04092 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Target-positive lysate and matched negative control (standard starting point) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | the SIX2 peptide (catalog A04092) |
| Primary antibody | A04092; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Species-matched HRP conjugate at validated dilution (standard starting point) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL; bracket exposures to avoid saturation (standard starting point) |
Section 2
What Is the Expected SIX2 Western Blot Band Size?
SIX2 is predicted at 32.3 kDa; no empirical band size is supplied, and its listed features do not establish altered migration.
| Single band near 32.3 kDa | Consistent with predicted SIX2 size; confirm its identity |
| Band near 32.3 kDa enriched in nuclear extract | Consistent with SIX2 nuclear localization |
| Faint band near 32.3 kDa in whole-cell lysate | Nuclear SIX2 may be diluted in the total sample |
| Little or no band near 32.3 kDa in a cytoplasmic fraction | Consistent with SIX2 nuclear localization |
💡Expected SIX2 appearanceSIX2 has a predicted mass of 32.3 kDa but no supplied empirical band size; use nuclear enrichment and peptide competition to assess a candidate band.
| Predicted molecular weight | Provides a 32.3 kDa reference for SIX2 |
| Predicted mass in daltons | Expresses the same reference as 32,286 Da |
| Predicted mass for band comparison | Supports checking a candidate band near 32.3 kDa |
| Predicted mass versus apparent migration | Does not establish where SIX2 runs experimentally |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear SIX2 may be diluted in whole-cell lysate | Check a nuclear extract and verify sample loading |
| Weak or no signal | SIX2 abundance in the tested sample is unknown | Check a nuclear extract and assay sensitivity |
| Band higher than expected | No annotated SIX2 feature establishes the shift | Test band identity by peptide competition |
| Band lower than expected | No annotated SIX2 cleavage explains a smaller band | Test band identity by peptide competition |
| Multiple bands | The supplied record lists only one isoform | Use peptide competition to identify the SIX2-reactive band |
Experiment evidence
Western blot images showing SIX2 band
A04092 · SIX2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SIX2 Western blot
🧪For positive controls for SIX2 in Western blot, you can use no HPA-validated tissue or cell line from the supplied evidence.
Positive control: No high/medium HPA tissue identified
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside.
⚠️Feasibility: HPA provides no tissue data, so suitable tissue controls cannot be identified here.
HPA tissue expression evidence for SIX2
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 |
|---|---|---|---|---|
| No high/medium HPA tissues identified in the supplied evidence. | ||||
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| No lower-expression tissue rows available in the supplied evidence. | ||||
Section 3
Advanced SIX2 Western Blot Tips
Deeper troubleshooting and optimisation questions for SIX2, answered from its protein features.
How should SIX2 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 SIX2 isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It provides no basis for assigning additional bands to annotated SIX2 isoforms.
Can annotated modifications explain a shifted SIX2 band?
PTM · No modified residues or glycosylation sites are listed. These features do not explain a shift, but their absence does not rule out unannotated modifications or establish where SIX2 will migrate.
Does this guide establish induction of SIX2?
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 SIX2?
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 A04092 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 SIX2 signals be quantified across samples?
Quantitation · Compare matched sample fractions with equal protein loading and signals within the linear detection range. Because SIX2 is nuclear, differences in nuclear recovery could affect measured intensity.
Where should SIX2 appear relative to its predicted mass?
Interpretation · SIX2 has a predicted mass of 32.3 kDa. No observed Western blot band is supplied, so its apparent mass cannot be compared with that prediction.
SIX2 is annotated as nuclear. Include a nuclear-enriched sample when assessing detection, and compare the same sample fraction across conditions.
SIX2 interacts with TCF7L2 and OSR1, but those interactions do not establish that an extra Western blot band contains SIX2. Confirm unexpected bands independently before assigning them to SIX2 or a complex.
SIX2 is annotated as nuclear. Include a nuclear-enriched sample when assessing detection, and compare the same sample fraction across conditions.
SIX2 interacts with TCF7L2 and OSR1, but those interactions do not establish that an extra Western blot band contains SIX2. Confirm unexpected bands independently before assigning them to SIX2 or a complex.
Boster reagents
SIX2 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-Homeobox protein SIX2 Six2 Antibody
Cat # A04092
A04092 is a SIX2 antibody listed for human and mouse reactivity. Its Western blot image includes a lane blocked with SIX2 peptide; the supplied caption does not identify the sample or blot conditions.
Which to pick: A04092 is the only listed option. It has a Western blot image with a peptide-blocked lane, but check suitability for your sample and protocol because those details are not supplied.
Source: BosterBio SIX2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9NPC8.
- Human Protein Atlas. SIX2 tissue expression.
- PMC5704498 — target-verified WB comparison
- PMC12170904 — target-verified WB comparison
- PMC6668977 — target-verified WB comparison