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- Table of Contents
TOX / Thymocyte selection-associated high mobility group box protein TOX · Western blot design guide
Design a Western Blot for TOX
Real validated TOX Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TOX 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
TOX 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 | ~57.5 kDa | |
| Observed band | ~57 kDa | |
| Gel | 5–20% (catalog A08441-2) | |
| Positive control | Appendix (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 | — | |
| Caveat | — | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated TOX Western Blot Protocols
The A08441-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Jurkat, human Ramos, human HL-60 (catalog A08441-2) |
| Gel % | 5–20% (catalog A08441-2) |
| Load | 30 ug; reducing conditions (catalog A08441-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A08441-2) |
| Membrane | nitrocellulose membrane (catalog A08441-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A08441-2) |
| Primary antibody | A08441-2 · 0.5 μg/mL (catalog A08441-2) |
| Primary incubation | overnight at 4°C (catalog A08441-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A08441-2) |
| Secondary incubation | 1.5 hour at RT (catalog A08441-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A08441-2) |
| Detection | ECL (catalog A08441-2) |
Section 2
What Is the Expected TOX Western Blot Band Size?
TOX is predicted at 57.5 kDa and observed near 57 kDa; the cause of the small difference is not established.
| Band near 57 kDa in whole-cell lysate | Matches the observed TOX band |
| Single band near 57 kDa | Consistent with the predicted 57.5 kDa mass |
| Band near 57 kDa in a nuclear fraction | Consistent with TOX's nuclear location |
| Little signal near 57 kDa in a cytoplasmic fraction | Consistent with TOX's nuclear location |
💡Expected TOX appearanceTOX has a predicted mass of 57.5 kDa and an observed band near 57 kDa; confirm band identity with appropriate controls.
| Predicted molecular mass of 57.5 kDa | Places the expected full-length band near 57.5 kDa |
| UniProt molecular weight of 57,513 Da | Corresponds to approximately 57.5 kDa |
| Full-length predicted mass | Provides the baseline for comparing other band positions |
| Predicted TOX protein size | Is consistent with the observed band near 57 kDa |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear TOX may be poorly recovered during extraction | Check extraction and probe a nuclear fraction |
| Band higher than expected | The supplied features do not establish a larger TOX form | Check band identity with an independent TOX antibody |
| Band lower than expected | The supplied features do not establish a smaller TOX form | Check band identity with an independent TOX antibody |
| Multiple bands | The supplied features do not establish distinct migrating TOX forms | Compare bands using an independent TOX antibody |
| Weak or no signal | Nuclear TOX may be poorly represented in the tested fraction | Check loading and probe a nuclear fraction |
Experiment evidence
Western blot images showing TOX band
A08441-2 · TOX
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TOX Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for TOX in Western blot, you can use appendix tissue, which HPA rates High for TOX.
Positive control: Appendix (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: TOX is nuclear, so a nuclear-enriched lysate may improve detection.
HPA tissue expression evidence for TOX
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 |
|---|---|---|---|---|
| Appendix | lymphoid tissue | High | Protein (IHC) | HPA → |
| Kidney | collecting ducts | High | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | High | Protein (IHC) | HPA → |
| Testis | sertoli cells | High | Protein (IHC) | HPA → |
| Tonsil | germinal center cells | High | 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 → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Epididymis | glandular cells | Not detected | Protein (IHC) | HPA → |
| Fallopian tube | glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TOX Western Blot Tips
Deeper troubleshooting and optimisation questions for TOX, answered from its protein features.
How should TOX 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 TOX isoforms explain additional bands?
Isoforms · The supplied UniProt features list one isoform and no alternative sequence. They provide no basis for assigning additional bands to TOX isoforms.
Do annotated modifications prove a band shift?
PTM · The linked UniProt record describes protein features. A modification annotation alone does not demonstrate a visible shift; retain any condition or experimental qualifier attached to it.
Does this guide establish induction of TOX?
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.
What transfer method to use for TOX Western blot?
Transfer · Plan transfer around the reported ~57 kDa band and check transfer at that size with a marker or total-protein stain. The supplied features do not specify a special transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A08441-2 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 TOX be quantified across samples?
Quantitation · Measure the ~57 kDa band in comparable samples or nuclear fractions. Use a consistent loading reference for the fraction analyzed; TOX's nuclear annotation makes fraction consistency particularly relevant.
How does the observed TOX band compare with its predicted mass?
Interpretation · TOX is predicted at 57.5 kDa, close to the reported ~57 kDa band. This agreement supports the expected band position, but mass alone does not establish identity.
TOX is annotated as nuclear. Include a nuclear fraction when checking for its ~57 kDa band, and compare like fractions across samples.
Check whether the ~57 kDa band is present before assigning other bands to TOX. The supplied features list no alternative sequence, modified residues, or glycosylation sites, so they do not establish an identity or cause for extra bands.
TOX is annotated as nuclear. Include a nuclear fraction when checking for its ~57 kDa band, and compare like fractions across samples.
Check whether the ~57 kDa band is present before assigning other bands to TOX. The supplied features list no alternative sequence, modified residues, or glycosylation sites, so they do not establish an identity or cause for extra bands.
Boster reagents
TOX 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-TOX Antibody Picoband®
Cat # A08441-2
The catalog reports one anti-TOX antibody, A08441-2, with reported Human and Mouse reactivity. Its Western blot image shows an approximately 57 kDa band in human Jurkat, Ramos, and HL-60 whole cell lysates. No mouse Western blot sample is shown.
Which to pick: A08441-2 is the only listed option. It has a Western blot image using human cell lysates at 30 µg per lane; Mouse reactivity is listed, but this image does not test a mouse sample.
Source: BosterBio TOX gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O94900.
- Human Protein Atlas. TOX tissue expression.
- PMC8062716 — target-verified WB comparison
- PMC7046285 — target-verified WB comparison