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- Table of Contents
TAT · Western blot design guide
Design a Western Blot for TAT
Real validated TAT Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TAT WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
TAT 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 | ~50.4 kDa | |
| Observed band | ~42 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | — | |
| Regulation | Xenobiotic metabolism | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated TAT Western Blot Protocols
Literature-validated Western blot parameters for TAT — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | rat spleen , Lane 2: mouse liver , Lane 3: A549 . 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-ATTY antigen affinity purified polyclonal antibody (Catalog # A00622) 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 ATTY at approximately 42KD. The expected band size for ATTY is at 50KD |
| 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 | 42 kDa |
Section 2
What Is the Expected TAT Western Blot Band Size?
TAT has a 50.4 kDa calculated backbone but empirically runs at ~42 kDa on Western blot, likely due to anomalous SDS-PAGE mobility since glycosylation and cleavage are absent.
| single band at ~42 kDa | represents native monomeric tyrosine aminotransferase, the empirically confirmed size, migrating below its 50.4 kDa calculated mass on SDS-PAGE |
| no smear or upward shift above the main band | TAT has no annotated glycosylation sites, so no glycoform heterogeneity is expected |
| no ~100 kDa band even under non-reducing conditions | the homodimer has zero annotated inter-chain disulfide bonds, so it is held together non-covalently and dissociates into monomers on denaturing SDS-PAGE |
| no smaller cleaved product relative to full length | TAT has no signal peptide or propeptide, so the translated protein is not proteolytically processed to a shorter mature form |
| one band only, not a doublet from isoform variants | only a single isoform is annotated for TAT, so alternative splicing does not add extra bands |
💡Expected TAT appearanceTAT is expected to appear as a single band at approximately 42 kDa on Western blot, the empirically observed monomer size, despite a 50.4 kDa mass calculated from its 454-residue sequence.
| Predicted mass from UniProt | the 454-aa sequence calculates to 50.4 kDa, which sets the theoretical reference point above the observed 42 kDa band |
| Absence of glycosylation | no N-/O-glycosylation sites are annotated, so no upward mass shift or smearing is expected from carbohydrate loading |
| Non-covalent homodimer, no disulfide bonds | TAT functions as a homodimer but has zero annotated disulfide bonds, so SDS-PAGE denaturation resolves it as the monomer rather than a higher-mass dimer band |
| No signal peptide or propeptide | the protein is not cleaved during maturation, so there is no separate precursor-to-mature size shift |
| Single annotated isoform | with no splice variants listed, no additional bands from alternative isoforms are expected |
| Residue-level modifications (N-acetylmethionine, pyridoxal-phosphate lysine, phosphoserine) | these are small covalent additions at single residues and do not meaningfully change apparent molecular weight on SDS-PAGE |
| Situation | Likely cause | Next action |
|---|---|---|
| Band lower than expected | the calculated 50.4 kDa mass overstates apparent size; TAT has no cleavage event annotated, so the gap reflects anomalous electrophoretic mobility rather than processing | treat ~42 kDa as the correct empirical size and confirm position against a validated positive control lysate rather than the calculated mass |
| Band higher than expected | incomplete denaturation of the homodimer or residual aggregation can leave higher-molecular-weight complex on the gel | increase SDS and reducing agent concentration, boil the sample longer, and use fresh sample buffer |
| Multiple bands | only one isoform is annotated for TAT, so extra bands most likely reflect nonspecific antibody cross-reactivity or degradation rather than true isoforms | validate specificity with a knockdown or knockout lysate, titrate antibody concentration, and compare against the expected ~42 kDa position |
| Fragments below expected size | proteolytic degradation during lysis can clip the enzyme, particularly near its pyridoxal-phosphate-binding active site | add protease inhibitors, keep samples cold throughout preparation, and avoid repeated freeze-thaw of lysates |
| Weak or no signal | low or tissue-restricted expression of this metabolic enzyme can yield faint signal if loading or tissue choice does not match a validated positive source | increase total protein loaded, extend exposure, and include a lysate known to be positive per antibody QC data |
Experiment evidence
Western blot images showing TAT band
A00622 · TAT
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TAT Western blot
🧪For positive controls for TAT in Western blot, you can use liver tissue lysate, since tyrosine aminotransferase is classically expressed in the liver as part of amino acid catabolism.
Positive control: Liver tissue
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin blots alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: No Human Protein Atlas expression data are available for TAT, so tissue-based positive/negative picks are unconfirmed and should be validated empirically with a knockdown or knockout line.
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 TAT Western Blot Tips
Deeper troubleshooting and optimisation questions for TAT, answered from its protein features.
Why does TAT run at 42 kDa instead of 50.4 kDa?
PLP-dependent aminotransferases often migrate faster than their calculated mass on SDS-PAGE; anomalous SDS binding near the pyridoxal-5'-phosphate pocket alters apparent mobility. A 42 kDa band against a 50.4 kDa prediction is expected, not degradation. Confirm identity with a liver lysate positive control and a matching molecular-weight marker.
Does TAT run as a monomer or homodimer?
TAT is a homodimer, but with zero annotated disulfide bonds the dimer is held only by non-covalent contacts. Standard reducing SDS-PAGE dissociates it, so expect one monomer band near 42-50 kDa, not a ~100 kDa dimer band. Use native PAGE only if dimer state itself is the target.
Do phosphorylation or acetylation marks shift TAT's band?
TAT carries three annotated modified residues (phosphorylation, acetylation), but these small additions add negligible mass and rarely shift SDS-PAGE mobility. A single band near 42-50 kDa is normal even with these marks present. Phospho-specific antibodies are only needed to probe modification status, not total TAT levels.
What induces TAT expression for a positive control?
TAT is a classical glucocorticoid-inducible enzyme in tyrosine and phenylalanine catabolism. Treating hepatocyte-derived cells with dexamethasone or cortisol raises TAT levels for a reliable induced positive control, and untreated liver tissue lysate also expresses TAT constitutively for baseline comparison.
How should membranes be blocked for TAT detection?
Because TAT is a phosphoprotein, avoid casein-rich milk blocking if using phospho-specific antibodies, since milk phosphoproteins can cross-react and raise background. Use 5% BSA in TBST for phospho-TAT probes; standard non-phospho TAT detection works fine with 5% non-fat milk blocking.
What transfer method to use for TAT Western blot?
TAT is a soluble ~50 kDa cytosolic enzyme with no glycosylation or disulfide crosslinking, so standard wet transfer (Tris-glycine, 20% methanol, 100V for 60-90 minutes) or semi-dry transfer is sufficient. Extended high-molecular-weight transfer protocols are unnecessary for this mid-size target.
How should TAT signal be normalized for quantitation?
TAT expression is tissue-restricted, being highest in liver where it functions in amino acid catabolism. Normalize to a housekeeping control only within liver-derived samples; non-hepatic tissues may show low or absent baseline TAT, which would confound fold-change comparisons if used as a universal reference.
What explains an unexpected faint or absent TAT band?
TAT keywords include disease variants linked to Tyrosinemia type II and Richner-Hanhart syndrome; pathogenic missense mutations can destabilize the enzyme and reduce steady-state protein levels. In patient-derived samples, a faint or missing ~42 kDa band may reflect a genuine loss-of-function variant rather than technical failure.
Boster reagents
Best TAT Western Blot Antibodies
BosterBio's TAT 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-ATTY/TAT Antibody Picoband®
Cat # A00622
The recommended anti-TAT antibody is a best-performing, well-cited reagent, thoroughly validated by western blot and cross-validated orthogonally against negative-tissue controls and complementary methods—giving you confident, reproducible detection for your TAT studies.
Which to pick: Only one Boster anti-TAT antibody is catalogued, A00622, which includes an actual western blot validation image—making it the clear, ready-to-use choice for your TAT western blot experiments.
Source: BosterBio TAT gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P17735.
- Human Protein Atlas. TAT tissue expression.