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TET1 · Western blot design guide
Design a Western Blot for TET1
Real validated TET1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TET1 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
TET1 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 | ~235.3 kDa | |
| Observed band | ~235 kDa | |
| Gel | 4–12% gradient | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Ubiquitinated | |
| Caveat | Multiple splice isoforms | |
| Regulation | LPS-induced | |
| Isoform | 4 isoform(s) |
Section 1
Real Curated TET1 Western Blot Protocols
Literature-validated Western blot parameters for TET1 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human HEK293 , Lane 2: human K562 , Lane 3: human SW620 , Lane 4: human HepG2 , Lane 5: monkey Cos-7 , Lane 6: human Raji . 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-TET1 antigen affinity purified polyclonal antibody (Catalog # A00603-2) at 0.25 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 TET1 at approximately 235KD. The expected band size for TET1 is at 235KD |
| Gel % | 4–12% gradient |
| Load | 50ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.25 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 | 235 kDa |
Section 2
What Is the Expected TET1 Western Blot Band Size?
TET1 has a predicted mass of ~235 kDa and runs at the same ~235 kDa observed empirically, since it lacks glycosylation, disulfide bonds, or proteolytic cleavage.
| single sharp band at ~235 kDa | matches the predicted full-length TET1 mass and the empirically observed native band, with no glycosylation or cleavage shifting it |
| faint lower-molecular-weight bands or a smeared trail below ~235 kDa | likely proteolytic degradation fragments, since a 235 kDa/2136-aa nuclear protein is highly susceptible to partial breakdown during lysis |
| additional distinct band(s) at sizes other than ~235 kDa | may correspond to one of the other three annotated TET1 splice isoforms rather than a nonspecific or degraded species |
| band position unchanged between reducing and non-reducing conditions | consistent with TET1 having no annotated inter-chain disulfide bonds, so no dimer-sized species is expected |
| signal strong in nuclear/chromatin-enriched fractions but weak or absent in cytoplasmic-only fractions | reflects TET1's annotated nuclear and chromosome/chromatin localization |
💡Expected TET1 appearanceTET1 runs as a single band at approximately 235 kDa on reducing SDS-PAGE, matching its predicted full-length mass since it has no glycosylation, disulfide-linked dimerization, or signal-peptide cleavage to shift its apparent size.
| Predicted molecular weight (235.3 kDa, 2136 aa) | sets the expected native full-length band position; empirical antibody QC bands (~235 kDa) closely match this predicted mass |
| Four annotated splice isoforms (1, 2, 3, 4) | isoforms 2-4 can appear as additional bands distinct from the canonical ~235 kDa isoform 1 band, so extra bands are not necessarily nonspecific |
| Absence of annotated inter-chain disulfide bonds | predicts only the monomeric ~235 kDa band under both reducing and non-reducing conditions, with no higher dimer-sized species |
| No annotated signal peptide or propeptide | TET1 is made as a single mature polypeptide with no proteolytic maturation step, so the full-length band represents the mature protein directly rather than a cleaved precursor |
| Very large protein size (235 kDa/2136 aa) | migrates slowly and can resolve poorly on standard gels, and is more prone to partial proteolysis during lysis than smaller proteins |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | TET1 is nuclear/chromatin-associated and its extraction from whole-cell lysate can be inefficient | use a nuclear or chromatin-enriched extraction protocol, load more total protein, and confirm the cell line expresses TET1 |
| Multiple bands | four annotated splice isoforms plus possible degradation products can generate several distinct sizes | compare band sizes against known isoform patterns and confirm specificity with a TET1 knockdown or knockout lysate control |
| Fragments below expected size | the large ~235 kDa TET1 protein is highly susceptible to proteolytic cleavage during sample preparation | prepare lysates fresh and cold with protease inhibitors, and avoid repeated freeze-thaw of lysates |
| Broad smear instead of sharp band | incomplete resolution of such a large protein on a standard percentage gel, or overloading of nuclear lysate | run on a low-percentage (5-6%) or gradient gel, reduce protein loading, and extend transfer time for the large protein |
| Weak or no signal | TET1 expression is restricted to certain cell types and the protein is chromatin-bound, limiting solubilization | use a cell line with confirmed TET1 expression as a positive control, enrich the chromatin fraction, and increase loading |
Experiment evidence
Western blot images showing TET1 band
A00603-2 · TET1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TET1 Western blot
🧪For positive controls for TET1 in Western blot, you can use lysate from TET1-overexpressing cells (e.g., HEK293T cells transiently transfected with a TET1 expression construct), since no tissue- or cell-line-specific expression data is available from the Human Protein Atlas.
Positive control: TET1-transfected HEK293T cells
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin alongside a total-protein stain (stain-free gel, Ponceau S, or REVERT) to confirm equal loading.
⚠️Feasibility: No HPA expression data exists for TET1, and as a nuclear, chromatin-associated protein it may be low-abundance in standard whole-cell lysates, so nuclear extraction plus an overexpression or siRNA/KO system is recommended to confirm 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 TET1 Western Blot Tips
Deeper troubleshooting and optimisation questions for TET1, answered from its protein features.
Why does TET1 run at its predicted molecular weight?
TET1 has a predicted mass of 235.3 kDa, no signal peptide or propeptide, and no annotated proteolytic maturation. The observed band at ~235 kDa matches this directly. Because no cleavage event is documented, a strong match between predicted and observed mass is expected; bands well below 235 kDa more likely reflect degradation of this large protein or a shorter isoform than altered processing.
Could multiple TET1 isoforms appear on the blot?
UniProt annotates four TET1 isoforms generated by alternative splicing and alternative promoter usage. An antibody raised against a region shared across isoforms can detect more than one species, producing extra bands smaller than 235 kDa. Check whether your antibody's epitope falls within a region present in all isoforms before treating secondary bands as nonspecific.
What regulates TET1 chromatin localization by ubiquitination?
TET1 carries an annotated modified residue, and its chromatin association is promoted by monoubiquitination at Lys-1589 rather than by proteolytic processing. Signal differences between nuclear soluble and chromatin-bound fractions can reflect this ubiquitination-dependent localization, so keep fraction identity in mind when comparing band intensities across sample types.
How should blocking be optimized for TET1 detection?
TET1 has no annotated disulfide bonds or glycosylation on this entry, so epitope accessibility is not complicated by oxidation state or sugar modification. Standard 5% non-fat milk or BSA in TBST is typically adequate; no specialized reducing or deglycosylation step is needed beyond routine denaturing SDS-PAGE sample preparation.
What transfer method to use for TET1 Western blot?
At 235 kDa, TET1 transfers poorly with semi-dry systems. Use wet (tank) transfer with a low-percentage (6-8%) acrylamide gel, and extend transfer time (e.g., low voltage overnight, or added SDS in transfer buffer) to move this large nuclear protein efficiently onto the membrane.
How should TET1 signal be quantified reliably?
Since TET1 is large and nuclear-restricted, use total protein normalization rather than a single small housekeeping control, and ensure adequate loading of nuclear or chromatin-enriched lysate. Only one modified residue is annotated, so isoform identity, not PTM heterogeneity, is the main factor to control for consistent quantitation.
What explains bands below 235 kDa for TET1?
Bands smaller than full-length 235 kDa most plausibly represent one of the four annotated splice or promoter-driven isoforms, or partial degradation of this very large protein during lysis. With no disulfide bonds or extensive glycosylation annotated, isoform diversity and proteolysis are more likely explanations than modification-driven mobility shifts.
Boster reagents
Best TET1 Western Blot Antibodies
BosterBio's TET1 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-TET1 Antibody Picoband®
Cat # A00603-2
Our recommended anti-TET1 antibodies are top-performing reagents, extensively cited in the literature and rigorously validated for Western blot, with specificity confirmed through orthogonal approaches including negative-tissue controls and complementary detection methods, ensuring reliable, reproducible TET1 detection.
Which to pick: Only one Boster TET1 antibody is catalogued, A00603-2, which includes an actual Western blot validation image—making it the clear, well-documented choice for your TET1 WB experiments.
Source: BosterBio TET1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q8NFU7.
- Human Protein Atlas. TET1 tissue expression.