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TRAK2 / Trafficking kinesin-binding protein 2 · IHC design guide
Design Immunohistochemistry for TRAK2
Plan chromogenic TRAK2 IHC in paraffin sections using cytoplasmic staining as the tissue benchmark (HPA tissue IHC). The IHC-validated antibody has a 1:100–1:300 dilution range (datasheet A07746); interpret staining cautiously because its consistency with RNA expression is low (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
TRAK2 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining across many tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A07746) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Hippocampus+2 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific) | |
| Caveat | Staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Staining intensity varies by tissue (HPA tissue IHC) | |
| Isoform / epitope | Two isoforms; check antibody epitope coverage (UniProt) |
Section 1
Recommended TRAK2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet A07746) is accompanied by one published TRAK2 IHC protocol for ESCC and corresponding para-cancer tissues (PMC12125856).
| Sample | Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A07746) |
| Fixation | Image fixative and duration unreported (datasheet A07746); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 9.0 (datasheet A07746); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-TRAK2, 1:100-1:300 (datasheet A07746) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | TRAK2-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated Tris-EDTA retrieval at pH 9.0 for the catalog antibody (datasheet A07746); the published ESCC protocol uses heated citrate buffer (PMC12125856: IHC methods).
Section 2
What Is the Expected TRAK2 Staining Pattern?
TRAK2 IHC is reported mainly as cytoplasmic staining across tissues, including strong staining in several glandular and epithelial cell populations (HPA tissue IHC). Cytoplasmic, early endosomal and mitochondrial localization is annotated, and TRAK2 has no transmembrane segment (UniProt O60296). Interpret a positive slide cautiously: the HPA tissue IHC profile is rated Uncertain because antibody staining and RNA expression have low consistency (HPA tissue IHC).
| Strong cytoplasmic staining in appendix glandular cells (HPA: High in appendix glandular cells). | This fits a reported positive IHC pattern. Assess the cell compartment and tissue structure together; staining intensity alone cannot establish antibody specificity because tissue IHC reliability is Uncertain (HPA tissue IHC). |
| Chromogen chiefly outlines cell membranes or fills extracellular space, with little cytoplasmic signal. | That distribution does not fit the reported cytoplasmic tissue pattern or the absence of a transmembrane segment (HPA tissue IHC; UniProt O60296 topology). Consider nonspecific staining or a slide artefact and check a control section. |
| Strong staining in smooth muscle cells, while expected positive epithelial cells remain faint. | Smooth muscle cells are reported as Not detected, so this pattern raises concern for cross-reactivity or endogenous detection activity (HPA tissue IHC). Compare matched controls before assigning the signal to TRAK2. |
| Diffuse chromogen obscures cell boundaries in both the specimen and its negative control. | Background prevents compartment-based interpretation. Check blocking, washing and the detection control as general IHC practice; the HPA tissue profile cannot establish the cause of diffuse background (HPA tissue IHC). |
| Appendix glandular cells show no convincing cytoplasmic signal (HPA: High in appendix glandular cells). | A failed positive control, an unsuitable antibody or an ineffective IHC workflow is possible. Review the catalog antibody's validated conditions and repeat with controls; one negative slide does not overturn an Uncertain tissue profile (HPA tissue IHC). |
💡Expected TRAK2 appearanceCall an appendix section positive when glandular cells show clear, strong cytoplasmic chromogen (HPA: High in appendix glandular cells); diffuse extracellular staining or dominant membrane outlining warrants investigation because the reported tissue pattern is cytoplasmic (HPA tissue IHC).
| Tissue and cell selection | Several glandular and epithelial populations stain High, whereas smooth muscle cells are Not detected (HPA tissue IHC). Choose controls by named cell type, since adjacent cells need not share the same reported level. |
| Strength of IHC evidence | The tissue profile is Uncertain, and antibody HPA015827 has an Uncertain IHC designation (HPA tissue IHC; HPA antibodies). Treat agreement with the map as supportive, not definitive verification of specificity. |
| Compartment evidence across applications | Tissue IHC reports mainly cytoplasmic signal, while ICC-IF reports approved nucleoplasm and nuclear bodies, plus vesicles (HPA tissue IHC; HPA subcellular). Record the assay used when interpreting a compartment mismatch. |
| Topology and processing | TRAK2 has no transmembrane segment or annotated signal peptide and is listed as a single chain spanning residues 1–914 (UniProt O60296). This supports assessing intracellular staining; it does not establish an antibody epitope. |
| Isoforms and epitope coverage | Two isoforms are listed, but antibody epitope coverage is not supplied (UniProt O60296; HPA antibodies). Do not attribute a staining difference to one isoform without independent evidence. |
| Detection chemistry | Endogenous enzyme activity or nonspecific detector binding can mimic chromogen signal (general IHC practice). Use appropriate detection controls; neither HPA staining levels nor UniProt annotations identify the cause on a particular slide. |
| Situation | Likely cause | Next action |
|---|---|---|
| No cytoplasmic signal in appendix glandular cells (HPA: High in appendix glandular cells). | The positive control or staining workflow may have failed; tissue IHC reliability is Uncertain (HPA tissue IHC). | Check section quality, retrieval and the catalog antibody's IHC-P conditions; repeat alongside detection controls (general IHC practice). |
| Smooth muscle cells stain strongly (HPA: Not detected in smooth muscle cells). | Cross-reactivity or endogenous detection activity is possible; a single discordant stain does not identify which (HPA tissue IHC; general IHC practice). | Inspect morphology and a control omitting primary antibody; compare with a reported positive cell type in the same run (HPA tissue IHC; general IHC practice). |
| Background is widespread, including areas without intact cells. | Nonspecific reagent binding, insufficient washing or endogenous detection activity may contribute (general IHC practice). | Review blocking, wash steps and detector-only controls before scoring cellular intensity (general IHC practice). |
| Chromogen is chiefly extracellular or sharply membrane outlining. | This conflicts with the mainly cytoplasmic tissue IHC profile and no transmembrane segment is annotated (HPA tissue IHC; UniProt O60296). | Recheck tissue morphology and detection controls, then compare intracellular staining in a reported positive cell type (HPA tissue IHC; general IHC practice). |
| IHC appears mainly nuclear rather than cytoplasmic. | Tissue IHC reports cytoplasm, but ICC-IF reports approved nuclear locations; the assays give different compartment evidence (HPA tissue IHC; HPA subcellular). | Document the discordance and verify IHC staining with controls; do not equate an ICC-IF localization with validated paraffin-section IHC (HPA tissue IHC; HPA subcellular). |
| What should nuclear or vesicular TRAK2 signal in ICC-IF mean? | ICC-IF reports nucleoplasm and nuclear bodies as main locations and vesicles as an additional location (HPA subcellular). | Interpret that result within the separate IF/ICC workflow; antibody HPA062163 is ICC Approved, while the tissue IHC profile is Uncertain (HPA antibodies; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing TRAK2 staining
A07746 · IHCImmunohistochemical analysis of paraffin-embedded human tonsil. 1, Antibody was diluted at 1:200 (4° overnight). 2, Tris-EDTA, pH9.0 was use
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000115993-TRAK2/tissue/cerebral+cortex#imid_4181884
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000115993-TRAK2/tissue/colon#imid_4181810
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000115993-TRAK2/tissue/liver#imid_4181795
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000115993-TRAK2/tissue/kidney#imid_4181796
![HPA ICC-IF BJ [Human fibroblast] — TRAK2 (HPA062163)](https://images.proteinatlas.org/62163/1368_F2_6_blue_red_green.jpg)
HPA · ICC-IF BJ [Human fibroblast]Human Protein Atlas immunofluorescence, BJ [Human fibroblast], antibody HPA062163; green = TRAK2, blue = nucleus, red = microtubules.
![HPA ICC-IF BJ [Human fibroblast] — TRAK2 (HPA062163)](https://images.proteinatlas.org/62163/1368_F2_7_blue_red_green.jpg)
HPA · ICC-IF BJ [Human fibroblast]Human Protein Atlas immunofluorescence, BJ [Human fibroblast], antibody HPA062163; green = TRAK2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for TRAK2 IHC & IF
🧪Run bronchus first: respiratory epithelial cells should stain strongly (HPA: High in bronchus respiratory epithelial cells). Use ovary as the negative tissue, scoring ovarian stroma cells for background staining (HPA: Not detected in ovary ovarian stroma cells); on the bronchus slide, use unstained adjacent cells as an internal background reference, without assuming they are TRAK2-negative.
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Hippocampus (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show TRAK2 in BJ [Human fibroblast], PC-3, U2OS, with annotated localisation: Nucleoplasm (approved), Nuclear bodies (approved) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, an isotype-matched control appropriate to the primary antibody’s host species and clonality, and a TRAK2 knockout specimen or validated peptide-block control (standard IHC practice). Check the bronchus slide for endogenous peroxidase activity before chromogenic detection; block endogenous biotin if using avidin–biotin detection (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the fixative in the selected A07746 paraffin-section tonsil caption is unreported (selected A07746 caption). That caption uses Tris–EDTA at pH 9.0 for retrieval and a 1:200 primary dilution, but does not establish retrieval dependence in bronchus (selected A07746 caption). There is insufficient supplied evidence to say frozen sections or IF are easier; assess bronchial background alongside the positive respiratory epithelium (HPA: High in bronchus respiratory epithelial cells; standard IHC practice).
HPA tissue IHC evidence for TRAK2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low consistency between antibody staining and RNA expression data. Pending external verification.). 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cervix | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Endothelial cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
Section 3
Advanced TRAK2 IHC Tips
Use the documented paraffin section conditions as a starting point, then assess staining by cell type and compartment (datasheet A07746; HPA tissue IHC).
Which retrieval should I try first for TRAK2 in paraffin sections?
Start with heat-mediated Tris-EDTA at pH 9.0 for TRAK2 staining in paraffin sections (datasheet A07746). Pair this with the documented 1:200 primary incubation at 4 °C overnight before judging retrieval failure (datasheet A07746). Run matched sections with and without retrieval, keeping section thickness, primary dilution, chromogen development, and counterstain identical (standard IHC practice). Because TRAK2 lacks a transmembrane segment and has cytoplasmic, early-endosome, and mitochondrial annotations, score cellular staining by compartment (UniProt O60296). If staining stays weak, titrate heating duration on paired sections before testing an alternative buffer, while monitoring morphology and background (standard IHC practice).
Does the tonsil IHC image establish which fixation preserves TRAK2?
The tonsil image documents paraffin sections but does not state a fixative, so TRAK2-specific fixation sensitivity remains unknown (datasheet A07746). For newly prepared specimens, record fixative, tissue thickness, and actual time in fixative; standardize these variables across cases (standard IHC practice). If routine 10% neutral buffered formalin is used, compare matched sections after the same Tris-EDTA pH 9.0 retrieval and 1:200 primary incubation (standard IHC practice; datasheet A07746). Examine loss of cellular signal alongside tissue architecture, rather than assigning a TRAK2-specific fixation mechanism from its topology or phosphorylation (UniProt O60296; standard IHC practice).
How should I interpret cytoplasmic and nuclear TRAK2 staining?
For tissue IHC, score cytoplasmic staining separately because that is the reported pattern in most tissues (HPA tissue IHC). TRAK2 also has cytoplasmic, early-endosome, and mitochondrial annotations, including MGARP-dependent mitochondrial recruitment reported by similarity (UniProt O60296). Nuclear staining needs separate scrutiny: nucleoplasm and nuclear bodies are approved locations in the subcellular dataset, while vesicles are an additional location (HPA subcellular). Compare compartment patterns across adjacent cells and matched controls under identical pH 9.0 retrieval and chromogen development (datasheet A07746; standard IHC practice). These sources describe different assays, so a nuclear IHC pattern alone does not establish its specificity (HPA tissue IHC; HPA subcellular; standard IHC practice).
Can this IHC result distinguish TRAK2 isoforms or phosphorylation states?
TRAK2 has two listed isoforms, but the supplied antibody caption does not map its epitope or establish isoform selectivity (UniProt O60296; datasheet A07746). An IHC-positive cell therefore supports antigen recognition under the tested conditions, not assignment to isoform 1 or 2 (datasheet A07746; standard IHC practice). The protein contains a HAP1 N-terminal region at residues 48–353 and a recorded phosphoserine at 420; neither identifies this antibody’s binding site (UniProt O60296; datasheet A07746). Ask for an epitope map or compare independently validated reagents before making isoform or modification claims, using matched retrieval and scoring conditions (standard IHC practice).
How can IF help check the cell type and compartment seen by IHC?
Use IF as a separate check of cell identity and compartment; the tissue IHC caption supplies no IF staining conditions (datasheet A07746). In bronchus, multiplex TRAK2 with an epithelial marker to test whether labeled cells match the reported respiratory epithelial population (HPA tissue IHC; standard IF practice). Choose a fluorophore channel after checking tissue autofluorescence with an unstained section, and include single-color controls for spectral bleed-through (standard IF practice). TRAK2 has no transmembrane segment, but the antibody epitope is unmapped; optimize permeabilisation for access to an intracellular epitope without assuming a membrane-facing side (UniProt O60296; datasheet A07746; standard IF practice).
What should I change when TRAK2 chromogenic staining looks diffuse?
First compare a no-primary control with the stained section to separate detection-system background from primary-dependent color (standard IHC practice). The documented starting conditions are primary antibody at 1:200, 4 °C overnight, and secondary antibody at 1:200 for 30 minutes at room temperature (datasheet A07746). If background persists, titrate primary concentration and incubation while keeping Tris-EDTA pH 9.0 retrieval constant (datasheet A07746; standard IHC practice). Apply appropriate protein blocking, wash thoroughly, and block endogenous peroxidase before DAB detection as general chromogenic workflow steps (standard IHC practice). Check whether diffuse color follows tissue edges or damaged areas before calling it TRAK2 expression (standard IHC practice).
How should I quantify TRAK2 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, since cytoplasmic staining predominates in the tissue profile (HPA tissue IHC). For a specified population, report the percentage of positive cells and an H-score from 0–300 using intensity grades 0–3 (standard IHC practice). If counting positive cells per mm², normalize to evaluable tissue area and record excluded folds, necrosis, and edges (standard IHC practice). Keep retrieval at Tris-EDTA pH 9.0, antibody dilution, chromogen development, and image thresholds consistent across sections (datasheet A07746; standard IHC practice). Treat comparisons with HPA cell-level observations cautiously because their tissue staining reliability is uncertain (HPA tissue IHC).
Which findings argue that a TRAK2-positive IHC field is artefactual?
A cytoplasmic pattern across relevant cells is consistent with the tissue profile, but the HPA staining assessment is uncertain (HPA tissue IHC). High respiratory epithelial staining in bronchus and undetected smooth muscle cells are useful comparison patterns, not definitive controls (HPA tissue IHC). Question color confined to section edges, necrotic areas, or a no-primary control; these patterns warrant checks for processing artefact or endogenous enzyme activity (standard IHC practice). Purely nuclear IHC staining also needs independent review because the tissue profile is cytoplasmic, although nuclear locations are reported in a separate subcellular assay (HPA tissue IHC; HPA subcellular). Compare cell type, compartment, and controls before interpreting intensity as TRAK2 abundance (standard IHC practice).
Boster reagents
Best TRAK2 / Trafficking kinesin-binding protein 2 IHC Antibodies
A07746 is listed for human and rat IHC and IF (catalog: applications and reactivity); its IHC figure shows paraffin-embedded human tonsil (IHC image caption). No IF figure is supplied (catalog: IF image alts).
Real IHC data
Anti-GRIF-1 TRAK2 Antibody
Cat # A07746
A07746 will render with an IHC figure of paraffin-embedded human tonsil (IHC image caption). A07746 also lists IF and human and rat reactivity, but supplies no IF figure or rat tissue image (catalog: applications, reactivity, IF image alts, IHC image caption).
Which to pick: Choose A07746 for paraffin-section IHC: its human tonsil figure used 1:200 primary antibody and Tris-EDTA retrieval at pH 9.0; the fixative is unreported (IHC image caption). For IF, A07746 lists a 1:50 dilution, but supplies no IF figure or ICC application (catalog: IF dilution, IF image alts, applications). For human and rat work, A07746 lists both species; rat staining is not shown and clonality is unreported (catalog: reactivity, IHC image caption, clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O60296 (TRAK2_HUMAN, Trafficking kinesin-binding protein 2).
- Human Protein Atlas. TRAK2 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. TRAK2 subcellular location (ICC-IF): Mainly localized to the nucleoplasm and nuclear bodies. In addition localized to vesicles..
- Human Protein Atlas. TRAK2 antibody validation summary (2 antibodies).
- CircSLC22A3 inhibits the invasion and metastasis of ESCC via the miR-19b-3p/TRAK2 axis and by reducing the stability of m(6)A-modified ACSBG1 mRNA. BMC cancer 2025 — PMC12125856.
- PubMed PMID:11161814 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for TRAK2 (Trafficking kinesin-binding protein 2). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A07746). Validated IHC antibody: A07746 at 1:100-1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Hippocampus. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A07746); 2. Fixation: Image fixative and duration unreported (datasheet A07746); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: Tris-EDTA pH 9.0 (datasheet A07746); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-TRAK2, 1:100-1:300 (datasheet A07746); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Caveat: Staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: Two isoforms; check antibody epitope coverage (UniProt); Regulation: Staining intensity varies by tissue (HPA tissue IHC). Evidence: UniProt O60296, HPA tissue IHC · Uncertain, HPA ICC-IF · Mainly localized to the nucleoplasm and nuclear bodies. In addition…, Datasheet A07746, PMC12125856. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/TRAK2/immunohistochemistry-ihc-guide.