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- Table of Contents
HTT / Huntingtin · Western blot design guide
Design a Western Blot for HTT
Real validated HTT Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-HTT 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
HTT 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 | ~347.6 kDa | |
| Gel | 4–12% gradient (standard starting point) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Heart muscle (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated HTT Western Blot Protocols
The M00134 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | HeLa cell lysate (catalog M00134) |
| Gel % | 4–12% gradient (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Wet/tank transfer; optimize duration (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | M00134; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Species-matched HRP conjugate at validated dilution (standard starting point) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL; bracket exposures to avoid saturation (standard starting point) |
Section 2
What Is the Expected HTT Western Blot Band Size?
Full-length HTT is predicted at 347.6 kDa; listed modifications and repeat expansion could affect migration, but no empirical band or feature-specific shift is established.
| Band near 347.6 kDa | Consistent with full-length HTT; confirm identity with antibody controls |
| Band above 347.6 kDa | Could reflect repeat expansion; band identity and cause require verification |
| Close doublet near 347.6 kDa | Could reflect different phosphorylation states; separation is unproven |
| Slight mobility variation near 347.6 kDa | Could reflect acetylation; a visible effect is unproven |
💡Expected HTT appearanceUniProt predicts full-length HTT at 347.6 kDa; no empirical band size is supplied, so confirm any band near this position with antibody and sample controls.
| Predicted full-length mass | Sets the sequence-based reference at 347.6 kDa |
| Phosphoserine at residue 411 | May affect mobility, but no visible shift is established |
| N6-acetyllysine at residue 9 | May affect mobility, but no visible shift is established |
| Triplet repeat expansion | May change protein length; no variant mass or migration is supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Full-length HTT may have been poorly recovered or detected | Check lysate preparation and antibody performance with a positive-control lysate |
| Band higher than expected | Repeat expansion is possible, but the band's identity is unconfirmed | Verify HTT with a second antibody and assess repeat status if relevant |
| Band lower than expected | A fragment or unrelated band is possible; no cleavage site is supplied | Compare antibodies recognizing different HTT regions |
| Multiple bands | Different modification states are possible, but distinct bands are not established | Confirm band identity with a second antibody; test phosphatase sensitivity if appropriate |
| Weak or no signal | Insufficient HTT recovery or antibody detection is possible | Check loading, transfer, and a positive-control lysate |
Experiment evidence
Western blot images showing HTT band
M00134 · HTT
PA2268 · HTT
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for HTT Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for HTT in Western blot, you can use adrenal gland tissue, which HPA rates as high for HTT expression.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Heart muscle (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: HPA identifies a high-expression tissue and a not-detected tissue, making tissue controls feasible.
HPA tissue expression evidence for HTT
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 |
|---|---|---|---|---|
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Breast | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | Low | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Low | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
Section 3
Advanced HTT Western Blot Tips
Deeper troubleshooting and optimisation questions for HTT, answered from its protein features.
How should HTT 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.
Do the supplied features support multiple HTT isoforms?
Isoforms · Only one isoform is listed, with no alternative sequence. The supplied features therefore do not assign different bands to splice isoforms.
Which HTT acetylation sites matter when interpreting bands?
PTM · UniProt lists N6-acetyllysine at positions 9, 176, 234, 343, and 442. These are UniProt sequence coordinates; check the numbering convention of any antibody or paper before comparing sites. Their presence alone does not establish a detectable band shift.
UniProt lists phosphoserine at 411, 417, 419, 432, 640, 643, 1179, 1199, 1870, and 1874. CDK5 is specified for positions 1179 and 1199. Use UniProt coordinates when comparing site-specific results; phosphorylation alone does not prove a visible shift.
UniProt lists phosphoserine at 411, 417, 419, 432, 640, 643, 1179, 1199, 1870, and 1874. CDK5 is specified for positions 1179 and 1199. Use UniProt coordinates when comparing site-specific results; phosphorylation alone does not prove a visible shift.
Does this guide establish induction of HTT?
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 HTT Western blot?
Transfer · At a predicted 347.6 kDa, full-length HTT is large. Consider a transfer setup optimized for high-mass proteins, and check transfer efficiency before interpreting a weak or absent full-length band. The supplied features do not establish one universally effective transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M00134 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 I quantify HTT bands across samples?
Quantitation · Quantify the same clearly defined band across samples and report its apparent position alongside the 347.6 kDa predicted mass. HTT is listed in the cytoplasm and nucleus, so compare matched sample fractions. The supplied features do not provide an empirical band position or establish that every detected band represents full-length HTT.
What mass should I expect for full-length HTT?
Interpretation · The supplied sequence is 3,142 residues with a predicted mass of 347.6 kDa. No empirical apparent band position is supplied. Compare bands near this mass cautiously: the listed modifications and N-terminal glutamine repeat do not, by themselves, establish a visible shift.
First compare them with the predicted 347.6 kDa full-length protein and the antibody’s recognized region. The sequence contains an N-terminal glutamine repeat, and triplet repeat expansion is listed, but no repeat length for a sample is supplied. The listed modifications do not establish the identity or apparent mass of an unexpected band.
First compare them with the predicted 347.6 kDa full-length protein and the antibody’s recognized region. The sequence contains an N-terminal glutamine repeat, and triplet repeat expansion is listed, but no repeat length for a sample is supplied. The listed modifications do not establish the identity or apparent mass of an unexpected band.
Boster reagents
HTT 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-Huntingtin Monoclonal Antibody
Cat # M00134
Real WB data
Anti-Huntingtin/HTT Antibody Picoband®
Cat # PA2268
Two the supplier anti-HTT antibodies, M00134 and PA2268, list human, mouse, and rat reactivity and have Western blot images. The supplied evidence shows HeLa lysate for M00134 and HeLa and U87 lysates for PA2268; no publication evidence was supplied.
Which to pick: Both list the same species reactivity. Choose PA2268 if its documented HeLa and U87 whole-cell lysate conditions (0.5 µg/mL antibody, 40 µg lysate) fit your experiment; its reported band is 348 kDa. M00134 has a HeLa lysate image.
Source: BosterBio HTT gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P42858.
- Human Protein Atlas. HTT tissue expression.
- PMC3110280 — target-verified WB comparison
- PMC3383872 — target-verified WB comparison
- PMC3469570 — target-verified WB comparison