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SIX3 · Western blot design guide
Design a Western Blot for SIX3
Real validated SIX3 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SIX3 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
SIX3 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 | ~35.5 kDa | |
| Observed band | ~35 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | — | |
| Caveat | — | |
| Regulation | LPS-induced | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated SIX3 Western Blot Protocols
Literature-validated Western blot parameters for SIX3 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | rat brain , Lane 2: mouse brain . After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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-Six3 antigen affinity purified polyclonal antibody (Catalog # A02604) 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 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for Six3 at approximately 35KD. The expected band size for Six3 is at 35KD |
| Gel % | 10–12% |
| Load | 50ug |
| 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, 1:10000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 35 kDa |
Section 2
What Is the Expected SIX3 Western Blot Band Size?
SIX3 has a 35.5 kDa predicted mass and runs at the expected ~35 kDa on Western blots, with no glycosylation, disulfide bonds, or isoforms causing a shift.
| single sharp band at ~35 kDa | matches the predicted 35.5 kDa mass of full-length SIX3, with no annotated modification adding mass |
| no smear or ladder above the main band | SIX3 has no annotated glycosylation sites, so no glycoform-driven upward shift or smearing is expected |
| no additional band at roughly double the main band under non-reducing conditions | no inter-chain disulfide bond is annotated for SIX3, so it is not expected to form a covalent homodimer |
| band strongest in nuclear-enriched lysate, weaker in cytoplasmic-only fractions | SIX3 is annotated as a nuclear homeodomain transcription factor, so signal should concentrate in nuclear extracts |
| no smaller cleaved band beneath the main band | no signal peptide or propeptide is annotated, so SIX3 is not proteolytically processed to a shorter mature form |
| only one band, not a cluster of closely spaced bands | UniProt lists a single SIX3 isoform, so multiple splice-variant bands are not expected |
💡Expected SIX3 appearanceSIX3 is expected to run as a single sharp band at approximately 35 kDa, consistent with its 35.5 kDa predicted mass, since it has no glycosylation, disulfide dimerization, or cleavage to shift it.
| predicted mass (35.5 kDa) | sets the baseline migration, matching the observed ~35 kDa band with essentially no shift |
| no annotated glycosylation sites | no upward mass shift or heterogeneous glycoform smearing is expected |
| no annotated inter-chain disulfide bonds | SIX3 should run as a monomer near 35 kDa under both reducing and non-reducing conditions, without a higher dimer band |
| single annotated isoform | only one band is expected, since there is no alternative-splicing size variant to generate extra bands |
| no signal peptide or propeptide | SIX3 is not proteolytically processed, so no smaller cleaved fragment should appear relative to full-length protein |
| non-glycosylated bacterial recombinant standard | because SIX3 carries no native glycosylation, a bacterially expressed recombinant standard should still run at essentially the same size as native protein |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | standard whole-cell lysis may under-extract this nuclear, chromatin-associated homeodomain protein | use a nuclear-extraction or RIPA buffer with sonication and confirm nuclear fraction recovery with a marker like Lamin B1 |
| Band higher than expected | with no annotated glycosylation or disulfide-linked dimer, a higher band more likely reflects incomplete denaturation or SDS-resistant aggregation than a genuine modification | increase heat/SDS denaturation time, ensure fresh reducing agent, and include a negative control to rule out cross-reactivity |
| Multiple bands | since only one isoform and no PTM-driven variants are annotated, extra bands likely reflect antibody cross-reactivity with related SIX-family homeodomain proteins or nonspecific binding | validate specificity using SIX3 knockdown/knockout lysate or a blocking peptide and titrate antibody concentration |
| Weak or no signal | SIX3 is a developmentally restricted, low-abundance nuclear transcription factor and may be absent or scarce in many cell lines | use a tissue or cell line known to express SIX3, such as embryonic neural/eye tissue, and increase total protein loaded |
| Fragments below expected size | SIX3 has no annotated cleavage site, so smaller fragments most likely arise from proteolytic degradation during lysate preparation | keep samples cold, add fresh protease inhibitors, and minimize freeze-thaw cycles before loading |
Experiment evidence
Western blot images showing SIX3 band
A02604 · SIX3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SIX3 Western blot
🧪For positive controls for SIX3 in Western blot, you can use HEK293 (or similar) cells transiently transfected with a SIX3 expression construct, since the Human Protein Atlas provides no confirmed endogenous positive tissue for this protein.
Positive control: SIX3-transfected HEK293 cells
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin antibody blots alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: As a nuclear homeobox transcription factor with no Human Protein Atlas expression data available, a native tissue-based positive/negative control pair cannot be confirmed, so an overexpression/knockdown system is the more reliable option.
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 SIX3 Western Blot Tips
Deeper troubleshooting and optimisation questions for SIX3, answered from its protein features.
Why does SIX3 run close to its predicted 35 kDa mass?
UniProt lists a single isoform with no signal peptide, propeptide, glycosylation, disulfide bonds, or other modified residues, so there is no processing or added mass to shift migration. The observed ~35 kDa band matches the 35.5 kDa predicted mass closely, making this a reliable size to expect and to use for confirming antibody specificity.
What transfer method to use for SIX3 Western blot?
At ~35.5 kDa, SIX3 transfers efficiently with standard wet or semi-dry transfer onto 0.2 um PVDF or nitrocellulose using a Tris-glycine buffer with 20% methanol. No unusual transfer conditions are needed since the protein has no signal peptide, glycosylation, or large isoform variants that would otherwise complicate efficient membrane transfer.
How should blocking be optimized for a nuclear homeodomain protein?
SIX3 is a nuclear, DNA-binding transcription factor, so ensure lysis and extraction conditions fully solubilize chromatin-associated protein before blocking. Standard 5% non-fat milk or BSA in TBST is appropriate since no phosphorylation sites are annotated; incomplete nuclear lysis, not blocking chemistry, is the more likely cause of weak signal.
Does SIX3 protein level change with HDAC or MTA1 activity?
UniProt notes SIX3 interacts with MTA1 and HDAC2 to repress its own transcription, forming a negative feedback loop. Treating cells with HDAC inhibitors or altering MTA1 expression may therefore change SIX3 protein abundance, which is useful context when comparing SIX3 band intensity across treatment or knockdown conditions.
Why might unexpected bands appear near the SIX3 signal?
SIX3 belongs to the homeobox family and its listed disease variants (holoprosencephaly-associated) are point mutations, not size-altering truncations, so extra bands are unlikely to reflect known isoforms. Cross-reactivity with other homeodomain proteins sharing conserved homeobox sequence, or incomplete denaturation of nuclear complexes with EYA4, MTA1, or HDAC2, are more probable causes.
How to best quantitate nuclear SIX3 signal across samples?
Because SIX3 is annotated as strictly nuclear, normalize band intensity to a nuclear loading control such as Lamin B or Histone H3 rather than a whole-cell control like GAPDH or actin. This corrects for variable nuclear fractionation efficiency between samples and gives a more accurate comparison of SIX3 levels.
Should whole-cell lysate or nuclear extract be used for detection?
Given SIX3's exclusively nuclear localization, nuclear extraction or fractionation before loading improves sensitivity and reduces background from cytoplasmic proteins. Whole-cell lysate can still be used but may dilute the SIX3 signal relative to a nuclear-enriched preparation, especially in cell types with low SIX3 expression.
Boster reagents
Best SIX3 Western Blot Antibodies
BosterBio's SIX3 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-Six3 Antibody Picoband®
Cat # A02604
Our top-recommended anti-SIX3 antibody for Western blot is a proven, extensively cited reagent validated through rigorous orthogonal testing, including confirmed specificity against negative tissue controls and cross-validation by complementary methods, ensuring reliable, reproducible, high-confidence detection of SIX3.
Which to pick: Only one SIX3 antibody is catalogued: A02604. It has a documented WB validation image showing a specific Six3 band on SDS-PAGE, so it's the clear and only choice for Western blot work on this target.
Source: BosterBio SIX3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O95343.
- Human Protein Atlas. SIX3 tissue expression.