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AMH · Western blot design guide
Design a Western Blot for AMH
Real validated AMH Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-AMH WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
AMH 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 | ~59.2 kDa | |
| Observed band | ~65 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Glycosylation increases band size | |
| Regulation | Apical junction | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated AMH Western Blot Protocols
Literature-validated Western blot parameters for AMH — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | 293T , Lane 2: COLO320 . 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-AMH antigen affinity purified polyclonal antibody (Catalog # A00763) 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 AMH4 at approximately 65KD. The expected band size for AMH is at 59KD |
| 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 | 65 kDa |
Section 2
What Is the Expected AMH Western Blot Band Size?
AMH has a 59.2 kDa predicted backbone but typically runs near 65 kDa on reducing blots, mainly due to N-glycosylation at Asn64 and Asn329.
| single band near 65 kDa on a reducing gel | full-length glycosylated AMH monomer, running above the 59.2 kDa predicted backbone due to N-linked glycosylation |
| band near double that size (roughly 120-130 kDa) under non-reducing conditions | inter-chain disulfide bonds link two monomers into a homodimer that migrates near twice the monomer mass |
| broad or smeared band around 60-70 kDa rather than a crisp line | heterogeneous occupancy of the two N-linked glycosylation sites (Asn64, Asn329) produces a mix of closely spaced species |
| an additional smaller band well below the ~65 kDa main band | proteolytic separation of the propeptide (residues 25-451) from the C-terminal mature growth-factor domain yields a smaller cleaved fragment |
| little or no signal in whole-cell lysate | AMH is a secreted protein, so most of it leaves the cell rather than accumulating intracellularly |
💡Expected AMH appearanceExpect a single reducing-gel band at approximately 65 kDa, above the 59.2 kDa predicted monomer mass because of N-glycosylation at Asn64 and Asn329; a disulfide-linked homodimer near double this size can appear if reduction is incomplete.
| Predicted mass (UniProt) | the unmodified 560-residue precursor calculates to 59.2 kDa, the baseline before any post-translational changes |
| N-glycosylation at Asn64 and Asn329 | added carbohydrate mass shifts the apparent band upward from the 59.2 kDa baseline toward the ~65 kDa empirically observed size and can broaden it |
| Inter-chain disulfide bonds / homodimer formation | under non-reducing or incompletely reduced conditions, monomers link covalently into a homodimer running near twice the monomer's apparent mass |
| Signal peptide cleavage (residues 1-24) | removal of the 24-residue signal peptide during secretion trims a small amount of mass before the circulating precursor is seen |
| Propeptide region (residues 25-451) | proteolytic release of this propeptide from the C-terminal mature domain can generate a smaller fragment distinct from the larger propeptide-containing precursor band |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | AMH is a secreted protein with little intracellular retention | probe conditioned media or serum/plasma rather than relying solely on whole-cell lysate, or concentrate secreted media before loading |
| Band higher than expected | N-glycosylation at Asn64 and Asn329 adds carbohydrate mass above the 59.2 kDa backbone, or incomplete reduction leaves the disulfide-linked homodimer intact | treat a parallel sample with PNGase F to confirm the glycan contribution and ensure a fresh, effective reducing agent is used before loading |
| Band lower than expected | proteolytic degradation during lysis or sample handling trims mass below the ~65 kDa full-length band | add protease inhibitors, keep samples cold throughout, and minimize freeze-thaw cycles before loading |
| Broad smear instead of sharp band | heterogeneous occupancy and branching of the two N-linked glycans produces a mix of similarly sized species | run a longer or higher-percentage gel for better resolution and include a PNGase F-treated control lane |
| Multiple bands | the full-length propeptide-containing precursor and a smaller cleaved mature growth-factor fragment can both be present | check whether the antibody epitope lies in the propeptide or mature domain to interpret which band(s) it should detect |
| Fragments below expected size | physiological cleavage separates the propeptide (residues 25-451) from the smaller C-terminal mature domain | expect this smaller fragment as a normal processing product and select an antibody specific to the domain of interest |
Experiment evidence
Western blot images showing AMH band
A00763 · AMH
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for AMH Western blot
🧪For positive controls for AMH in Western blot, you can use recombinant AMH protein or lysate/conditioned medium from an AMH-overexpressing cell line, since no Human Protein Atlas tissue or cell line expression data are available for this protein.
Positive control: Recombinant AMH protein / overexpression lysate
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin antibody probes alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: As a secreted protein, AMH may give weak signal in whole-cell lysates, so use conditioned medium or concentrated supernatant, and note that with no HPA expression data available, no confirmed tissue-based positive/negative pair can be specified for this protein.
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 AMH Western Blot Tips
Deeper troubleshooting and optimisation questions for AMH, answered from its protein features.
Why does AMH run at ~65 kDa instead of 59.2 kDa predicted?
AMH's calculated mass is 59.2 kDa, but its 2 N-glycosylation sites add carbohydrate mass, shifting the observed band to ~65 kDa. Run reducing SDS-PAGE to eliminate mobility differences caused by the 8 disulfide bonds, which alter native folding and migration relative to the linear predicted sequence.
Why might multiple AMH bands appear on the blot?
AMH is synthesized as a proprotein (residues 25-451) that is proteolytically cleaved into N- and C-terminal fragments held together non-covalently. Under non-reducing conditions the disulfide-linked homodimer migrates near ~130 kDa. Expect uncleaved precursor, cleaved ~65 kDa monomer, and dimer bands depending on reduction and processing state.
Does AMH have isoforms that affect the band pattern?
UniProt lists only a single AMH isoform, so isoform-driven band multiplicity is not expected. Extra bands are more likely explained by proprotein processing (signal peptide and propeptide cleavage) or glycosylation heterogeneity rather than alternative splicing.
How should blocking be optimized for AMH detection?
Because AMH carries 2 N-glycosylation sites, milk-based blockers can raise background from lectin-like interactions with glycan structures. Use a BSA-based blocking buffer and extend blocking time to account for AMH's secreted, glycosylated nature, reducing nonspecific antibody binding around the target band.
What transfer method to use for AMH Western blot?
AMH is secreted as a disulfide-linked homodimer within a larger heterotetrameric complex, so higher-molecular-weight species transfer inefficiently with rapid methods. Use wet transfer with a PVDF membrane rather than semi-dry transfer to ensure both the ~65 kDa monomer and any dimeric forms transfer completely.
How should AMH be quantified across different samples?
Since AMH is a secreted protein, conditioned media or serum samples lack intracellular housekeeping proteins like GAPDH or actin for normalization. Quantify using total protein staining (e.g., Ponceau) or a fixed loading volume/concentration rather than relying on a cytoplasmic loading control.
What induces AMH expression in target tissues?
AMH functions as a growth factor in gonadal differentiation, consistent with its annotated role in this developmental pathway. Expression is tied to gonadal (e.g., Sertoli cell) identity rather than being broadly inducible. If probing non-gonadal or adult samples, confirm the tissue is expected to express AMH before interpreting a missing band.
Boster reagents
Best AMH Western Blot Antibodies
BosterBio's AMH 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-AMH Antibody Picoband®
Cat # A00763
Boster's anti-AMH antibody is a best-performing, highly cited reagent validated by SDS-PAGE Western blot and cross-checked against complementary methods and negative-control tissue, giving confident, reproducible detection of AMH protein across sample types.
Which to pick: Only one AMH antibody is catalogued, A00763, which includes an actual Western blot validation image—making it the clear choice for AMH WB experiments; no alternative SKUs are available to compare against.
Source: BosterBio AMH gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P03971.
- Human Protein Atlas. AMH tissue expression.