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SOX5 · Western blot design guide
Design a Western Blot for SOX5
Real validated SOX5 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SOX5 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
SOX5 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 | ~84 kDa | |
| Observed band | ~84 kDa | |
| Gel | 8–10% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Multiple splice isoforms | |
| Regulation | LPS-induced | |
| Isoform | 5 isoform(s) |
Section 1
Real Curated SOX5 Western Blot Protocols
Literature-validated Western blot parameters for SOX5 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | Rat Liver at 50ug, Lane 2: Rat Testis at 50ug, Lane 3: Rat Brain at 50ug, Lane 4: HELA at 40ug, Lane 5: A549 at 40ug. 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-SOX5 antigen affinity purified polyclonal antibody (Catalog # PB9507) 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 Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for SOX5 at approximately 84 kDa. The expected band size for SOX5 is at 84 kDa |
| Gel % | 8–10% |
| 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 | 84 kDa |
Section 2
What Is the Expected SOX5 Western Blot Band Size?
SOX5 runs as a single ~84 kDa band matching its predicted mass, with any higher or extra bands reflecting homodimer/SOX6 heterodimer formation or isoform variation rather than modification.
| single sharp band at ~84 kDa | matches SOX5's predicted monomeric mass, consistent with an unmodified, uncleaved core protein |
| band near ~168 kDa under non-reducing or incompletely denaturing conditions | reflects SOX5 homodimer formation or SOX5-SOX6 heterodimer complex that has not fully dissociated |
| multiple bands at slightly different molecular weights | consistent with expression of more than one of the five annotated SOX5 splice isoforms |
| signal concentrated in nuclear fraction, weak or absent in cytoplasmic-only fraction | expected because SOX5 is a nuclear transcription factor, not a cytoplasmic or secreted protein |
| slight smear or fine doublet around the main ~84 kDa band | can arise from heterogeneous phosphorylation across SOX5's multiple modified serine/threonine residues |
💡Expected SOX5 appearanceExpect a single sharp band at approximately 84 kDa in nuclear or whole-cell lysate, matching SOX5's predicted monomeric mass since it lacks a signal peptide, propeptide, glycosylation, or disulfide crosslinks.
| predicted monomer mass (84 kDa) | sets the baseline single-band position observed on most SOX5 blots, matching the empirical ~84 kDa band |
| homodimer and SOX5-SOX6 heterodimer formation | can produce a higher apparent band near double the monomer mass if dimers are not fully reduced or denatured |
| five annotated splice isoforms (1-5) | may generate additional bands of somewhat different sizes depending on which isoform(s) a tissue expresses |
| multi-site phosphorylation (7 phosphoserine/phosphothreonine residues) | can cause a minor upward mobility shift or slight smearing around the main band |
| absence of signal peptide, propeptide, and glycosylation | means no cleavage- or glycosylation-driven mass shift is expected; any deviation from ~84 kDa points to dimerization or isoform effects instead |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | SOX5 is nuclear-localized and chromatin-associated, so standard whole-cell lysis without adequate nuclear disruption may leave it insoluble | use a lysis buffer with nuclease/sonication or a dedicated nuclear extraction protocol to fully solubilize SOX5 |
| Band higher than expected | incomplete reduction or denaturation leaves SOX5 homodimers or SOX5-SOX6 heterodimers intact near ~168 kDa | boil samples fully in fresh reducing sample buffer with DTT or beta-mercaptoethanol before loading |
| Multiple bands | SOX5 has five known splice isoforms that can co-express and resolve at different apparent sizes | compare band pattern against the predicted ~84 kDa dominant isoform and note tissue-specific isoform expression rather than treating extra bands as nonspecific |
| Weak or no signal | SOX5 is a low-abundance nuclear transcription factor, so insufficient nuclear protein loading can under-represent it | increase total protein loaded and enrich for the nuclear fraction before probing |
| Fragments below expected size | the large 84 kDa nuclear protein is susceptible to proteolytic degradation during lysis if inadequately protected | add fresh protease inhibitors and keep lysates cold throughout preparation to prevent breakdown into smaller fragments |
| Broad smear instead of sharp band | heterogeneous phosphorylation across SOX5's multiple modified residues can broaden the band's mobility | run a higher-percentage gel for finer resolution or treat a parallel sample with phosphatase to sharpen the band |
Experiment evidence
Western blot images showing SOX5 band
PB9507 · SOX5
PB9507 · SOX5
PB9507 · SOX5
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SOX5 Western blot
🧪For positive controls for SOX5 in Western blot, you can use a SOX5-overexpression lysate (e.g., transiently transfected HEK293 cells), since no confirmed endogenous positive tissue is available in the supplied HPA data.
Positive control: SOX5-overexpressing HEK293 lysate
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Alongside SOX5, run GAPDH and β-actin as loading controls plus a total-protein stain such as stain-free imaging, Ponceau S, or REVERT.
⚠️Feasibility: As a nuclear transcription factor with no HPA tissue expression data supplied, a validated endogenous positive/negative tissue pair cannot be confirmed, so pairing an overexpression lysate with siRNA knockdown or a KO line is the most feasible way to establish antibody specificity.
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 SOX5 Western Blot Tips
Deeper troubleshooting and optimisation questions for SOX5, answered from its protein features.
Does SOX5 band size match its predicted mass?
Yes. SOX5's predicted mass is 84 kDa and the observed band is also ~84 kDa, so no significant shift from PTMs or unusual migration is expected. This close match makes ~84 kDa a reliable size marker for confirming correct target detection on your blot.
Why might multiple SOX5 bands appear on blots?
SOX5 has 5 annotated isoforms from alternative splicing, so antibodies targeting shared regions can detect multiple bands of different sizes depending on tissue or cell type. Confirm your antibody's immunogen region against isoform sequences to predict which variants it should detect.
Could phosphorylation shift the SOX5 band position?
SOX5 is a phosphoprotein with 7 reported modified residues. Phosphorylation can cause slight upward mobility shifts or smearing on SDS-PAGE. If band position varies between samples, consider phosphatase treatment as a control to confirm whether shifts are phosphorylation-dependent rather than isoform-related.
What transfer method to use for SOX5 Western blot?
At ~84 kDa, SOX5 transfers well with standard wet transfer, which handles mid-to-large nuclear proteins more reliably than semi-dry systems. Use extended transfer times (60-90 minutes) or overnight transfer at low voltage/4°C to ensure efficient elution of this nuclear transcription factor from the gel.
Why is blocking optimization important for SOX5 detection?
SOX5 is a nuclear DNA-binding transcription factor, typically low-abundance relative to cytoplasmic proteins. Use 5% non-fat milk or BSA blocking and optimize antibody dilution to reduce background, since faint nuclear factor bands are easily masked by high background from insufficient blocking or short incubation.
What loading control suits SOX5 quantitation?
Since SOX5 localizes to the nucleus, pair it with a nuclear loading control such as Lamin B1 or Histone H3 rather than a cytoplasmic marker like GAPDH, especially if using nuclear/cytoplasmic fractionation to enrich for this transcription factor before quantitation.
Could higher molecular weight bands indicate SOX5 dimers?
SOX5 forms homodimers and heterodimers with SOX6. Incomplete denaturation or insufficient reducing/boiling conditions could leave residual dimer complexes, appearing as higher-molecular-weight bands roughly double the ~84 kDa monomer. Ensure thorough sample boiling in reducing SDS-PAGE buffer to resolve monomeric SOX5 cleanly.
Boster reagents
Best SOX5 Western Blot Antibodies
BosterBio's SOX5 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-SOX5 Antibody Picoband®
Cat # PB9507
Our recommended anti-SOX5 antibody is a top-performing, well-cited reagent from Boster's catalog, rigorously validated by western blot and cross-checked against negative tissue controls and complementary detection methods to ensure specific, reproducible SOX5 detection.
Which to pick: Only one SOX5 antibody is catalogued here, PB9507, so it's the default choice. It includes a genuine western blot validation image (rat liver tissue), giving direct evidence of specific SOX5 detection before you order.
Source: BosterBio SOX5 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P35711.
- Human Protein Atlas. SOX5 tissue expression.