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- Table of Contents
AMT / Aminomethyltransferase, mitochondrial · Western blot design guide
Design a Western Blot for AMT
Real validated AMT Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-AMT 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
AMT 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 | ~43.9 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | — | |
| Caveat | — | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 4 isoform(s) |
Section 1
Real Curated AMT Western Blot Protocols
The A03217 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | extracts of various cell lines, (catalog A03217) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | Blocking buffer: 3% nonfat dry milk in TBST (catalog A03217) |
| Primary antibody | A03217 · 1:1000 (catalog A03217) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Goat Anti-Rabbit IgG, 1:10000 (catalog A03217) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL (catalog A03217) |
Section 2
What Is the Expected AMT Western Blot Band Size?
AMT is predicted at 43.9 kDa; four listed isoforms could affect band patterns, but their migration has not been demonstrated here.
| Band near 43.9 kDa | Candidate AMT based on the predicted mass; confirm identity with controls |
| Single sharp band | One detectable AMT species; the listed isoforms need not resolve separately |
| Several bands at different positions | Isoforms 1, 2, 3 and 4 are possible contributors; band identities require validation |
| Faint or absent band in whole-cell lysate | Mitochondrial AMT may be easier to detect after mitochondrial enrichment |
💡Expected AMT appearanceAMT has a predicted mass of 43.9 kDa, but no empirical band size is supplied; use a band near that size as a starting expectation and confirm its identity with controls.
| UniProt predicted mass | Places the reference expectation near 43.9 kDa |
| Splice isoform 1 | Its individual mass and migration are not supplied |
| Splice isoform 2 | May differ in size from other isoforms; its mass is not supplied |
| Splice isoform 3 | May differ in size from other isoforms; its mass is not supplied |
| Splice isoform 4 | May differ in size from other isoforms; its mass is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Mitochondrial AMT may be below detection in this lysate | Check a positive control and compare with a mitochondria-enriched fraction |
| Band higher than expected | An isoform is possible, but its migration is unknown | Confirm identity with an independent AMT antibody or AMT-depleted sample |
| Band lower than expected | An isoform is possible, but its migration is unknown | Check isoform expression and validate the band with an independent antibody |
| Multiple bands | Four splice isoforms are listed, without demonstrated band positions | Compare bands with an AMT-depleted sample before assigning isoforms |
| Weak or no signal | The sampled lysate may contain little detectable mitochondrial AMT | Verify loading and antibody performance with a positive control, then try mitochondrial enrichment |
Experiment evidence
Western blot images showing AMT band
A03217 · AMT
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for AMT Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for AMT in Western blot, you can use adrenal gland tissue, which has high HPA expression.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside AMT.
⚠️Feasibility: No Not-detected tissue is listed, so use siRNA knockdown or a KO line for a negative control.
HPA tissue expression evidence for AMT
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 → |
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | neuronal cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Low | Protein (IHC) | HPA → |
| Soft tissue | fibroblasts | Low | Protein (IHC) | HPA → |
| Breast | glandular cells | Medium | Protein (IHC) | HPA → |
| Hippocampus | neuronal cells | Medium | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced AMT Western Blot Tips
Deeper troubleshooting and optimisation questions for AMT, answered from its protein features.
How should AMT 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 AMT isoforms produce different bands?
Isoforms · UniProt lists four isoforms. Isoform 2 lacks residues 30–85, isoform 3 lacks residues 113–156, and isoform 4 replaces residues 380–403 with LPSGPCF. These sequence differences could affect band size or antibody recognition, but the features do not establish which isoforms appear on a blot.
Check the antibody epitope against the listed changes: residues 30–85 are missing in isoform 2, residues 113–156 in isoform 3, and residues 380–403 are replaced in isoform 4. An antibody targeting one of those regions may detect isoforms differently. These are UniProt coordinates; antibody documentation may use another numbering convention.
Check the antibody epitope against the listed changes: residues 30–85 are missing in isoform 2, residues 113–156 in isoform 3, and residues 380–403 are replaced in isoform 4. An antibody targeting one of those regions may detect isoforms differently. These are UniProt coordinates; antibody documentation may use another numbering convention.
Do listed modifications explain an AMT band shift?
PTM · No modified residues or glycosylation sites are listed, and the glycosylation count is zero. The supplied features therefore provide no specific modification to assign to a shifted band. They also provide no observed band position with which to compare the predicted 43.9 kDa mass.
Does this guide establish induction of AMT?
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.
How should transfer be checked for AMT?
Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A03217 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 AMT bands be chosen for quantitation?
Quantitation · Use a consistently detected band and verify that the antibody epitope is present in the isoform being measured. The four listed isoforms have different sequences, so combining distinct bands could mix isoform signals. The supplied features do not identify an observed band or establish which isoforms are expressed.
Should AMT migrate at exactly 43.9 kDa?
Interpretation · 43.9 kDa is the predicted mass. No observed band position is supplied, and the listed features do not establish an apparent mass or a visible shift. Use 43.9 kDa as a starting reference when assessing bands.
AMT is mitochondrial and carries a transit-peptide keyword. The supplied features give no cleavage site or mature-chain mass, so they cannot establish that transit-peptide processing explains a lower band. Assess any additional band alongside the documented isoform sequences and antibody epitope.
AMT is the T protein of the four-protein glycine cleavage system, which also contains P, L and H proteins. That system membership does not identify an unexpected band as AMT or a partner. Compare the band's apparent position with the predicted AMT mass and check whether the antibody recognizes the relevant AMT isoform sequence.
AMT is mitochondrial and carries a transit-peptide keyword. The supplied features give no cleavage site or mature-chain mass, so they cannot establish that transit-peptide processing explains a lower band. Assess any additional band alongside the documented isoform sequences and antibody epitope.
AMT is the T protein of the four-protein glycine cleavage system, which also contains P, L and H proteins. That system membership does not identify an unexpected band as AMT or a partner. Compare the band's apparent position with the predicted AMT mass and check whether the antibody recognizes the relevant AMT isoform sequence.
Boster reagents
AMT 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-AMT Antibody
Cat # A03217
A03217 is an anti-AMT antibody listed for human, mouse, and rat. Its Western blot image is described as using extracts from various cell lines at 1:1000, with 25 µg per lane. The caption does not identify the individual cell lines.
Which to pick: A03217 is the only listed option and has a Western blot image. Check its stated human, mouse, and rat reactivity against your sample; the image caption does not specify which cell lines were tested.
Source: BosterBio AMT gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P48728.
- Human Protein Atlas. AMT tissue expression.
- PMC10130911 — target-verified WB comparison