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- Table of Contents
AHCY / Adenosylhomocysteinase · Western blot design guide
Design a Western Blot for AHCY
Source-linked AHCY Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-AHCY 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
AHCY Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~47.7 kDa | |
| Observed band | ~45–48 kDa | |
| Gel | 10% (catalog A04236-1) | |
| Positive control | Epididymis (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked AHCY Western Blot Protocol Options
The A04236-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human HepG2, human A549, human Jurkat, rat liver, rat thymus, mouse liver (catalog A04236-1) |
| Gel % | 10% (catalog A04236-1) |
| Load | 30 ug; reducing conditions (catalog A04236-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A04236-1) |
| Membrane | nitrocellulose membrane (catalog A04236-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A04236-1) |
| Primary antibody | A04236-1 · 0.5 μg/ml (catalog A04236-1) |
| Primary incubation | overnight at 4°C (catalog A04236-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A04236-1) |
| Secondary incubation | 1.5 hour at RT (catalog A04236-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A04236-1) |
| Detection | ECL (catalog A04236-1) |
Section 2
What Is the Expected AHCY Western Blot Band Size?
AHCY is predicted at 47.7 kDa and observed at approximately 45–48 kDa; the cause of any difference is not established.
| Band at approximately 45–48 kDa | Empirical AHCY band in reducing whole-cell and tissue lysates; confirm identity with antibody controls |
| Single band near 47.7 kDa | Consistent with the predicted AHCY monomer mass |
| Several bands near the expected region | AHCY has isoforms 1 and 2, but distinct migration has not been demonstrated |
| Band in a cytoplasmic fraction | Consistent with the reported cytoplasmic location of AHCY |
💡Expected AHCY appearanceAHCY has a predicted mass of 47.7 kDa and an empirical band at approximately 45–48 kDa in reducing lysates; confirm band identity with appropriate antibody controls.
| Predicted AHCY monomer mass | Sets a 47.7 kDa reference for the band observed at approximately 45–48 kDa |
| Isoform 1 | Its apparent size relative to isoform 2 is not supplied |
| Isoform 2 | Its apparent size relative to isoform 1 is not supplied |
| Ser183 phosphorylation | Could affect migration, but no visible size shift is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | AHCY signal may be below detection or the antibody may not recognize the sample | Check loading and transfer, then compare with a positive lysate |
| Band higher than expected | The identity or migration of the higher band is unestablished | Check reduction and compare with the 45–48 kDa band using antibody controls |
| Band lower than expected | An isoform or sample degradation is possible, but neither explains a specific size here | Compare fresh lysate and confirm the band with an independent antibody |
| Multiple bands | Isoforms 1 and 2 are listed, but their migration is not supplied | Use isoform-specific evidence or an independent antibody to assign bands |
| Weak or no signal | Low detectable AHCY or an antibody or transfer issue | Check loading and transfer and include a positive lysate |
| Fragments below expected size | Sample degradation is possible | Prepare fresh lysate with protease inhibitors and confirm fragment identity |
Experiment evidence
Western blot images showing AHCY band
A04236-1 · AHCY
M04236 · AHCY
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for AHCY Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for AHCY in Western blot, you can use epididymis tissue lysate, where HPA reports high expression.
Positive control: Epididymis (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside.
⚠️Feasibility: A clean negative tissue is feasible because HPA reports AHCY as not detected in adipose tissue.
HPA tissue expression evidence for AHCY
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 |
|---|---|---|---|---|
| Epididymis | glandular cells | High | Protein (IHC) | HPA → |
| Kidney | cells in tubules | High | Protein (IHC) | HPA → |
| Skin | fibroblasts | High | Protein (IHC) | HPA → |
| Testis | cells in seminiferous ducts | High | Protein (IHC) | HPA → |
| Thyroid gland | glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Low | Protein (IHC) | HPA → |
| Hippocampus | glial cells | Low | Protein (IHC) | HPA → |
| Lung | alveolar cells | Low | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Low | Protein (IHC) | HPA → |
Section 3
Advanced AHCY Western Blot Tips
Deeper troubleshooting and optimisation questions for AHCY, answered from its protein features.
How should AHCY 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 AHCY isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. Isoform 2 lacks residues 1–28 of the canonical sequence, so it may migrate differently. Confirm isoform detection against the antibody’s binding region; the sequence difference alone does not establish which band is which.
Which AHCY modifications are documented?
PTM · In UniProt canonical coordinates, AHCY has N-acetylserine 2, phosphoserine 183, N6-(2-hydroxyisobutyryl)lysine 186, and phosphotyrosine 193. Isoform 2 lacks canonical residues 1–28, including serine 2. State the numbering convention when comparing these sites with antibody or paper annotations.
The listed phosphorylation and other modifications identify possible sources of molecular variation, but their presence does not demonstrate a visible Western blot shift. Compare treated and control samples under the same conditions before attributing a mobility change to a modification.
The listed phosphorylation and other modifications identify possible sources of molecular variation, but their presence does not demonstrate a visible Western blot shift. Compare treated and control samples under the same conditions before attributing a mobility change to a modification.
Does this guide establish induction of AHCY?
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 AHCY?
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 A04236-1 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 AHCY in cellular fractions?
Quantitation · AHCY is reported in the cytoplasm, nucleus, endoplasmic reticulum, and melanosomes. Quantify comparable fractions with appropriate loading controls; a change in one fraction alone does not establish a change in total AHCY.
Does the observed AHCY band match its predicted mass?
Interpretation · AHCY is predicted at 47.7 kDa, and the reported Western blot band is approximately 45–48 kDa. The supplied features do not establish why apparent and calculated masses might differ.
Check whether a lower band is consistent with isoform 2, which lacks canonical residues 1–28. AHCY is also reported as a homotetramer, but that feature alone does not identify a higher band. Verify unexpected bands with an independent AHCY-specific check before assigning them.
Check whether a lower band is consistent with isoform 2, which lacks canonical residues 1–28. AHCY is also reported as a homotetramer, but that feature alone does not identify a higher band. Verify unexpected bands with an independent AHCY-specific check before assigning them.
Boster reagents
AHCY 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-AHCY Antibody Picoband®
Cat # A04236-1
Real WB data
Anti-AHCY Rabbit Monoclonal Antibody
Cat # M04236
Both listed anti-AHCY antibodies have Western blot images. A04236-1 shows a 45–48 kDa band in the specified human cell and rodent tissue lysates; M04236 shows AHCY expression in A549 lysate, with limited experimental detail provided.
Which to pick: Choose A04236-1 if you want documented WB conditions and examples spanning human, rat, and mouse samples. M04236 has a WB image from A549 lysate, but its caption provides fewer details. Both list human, mouse, and rat reactivity.
Source: BosterBio AHCY gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P23526.
- Human Protein Atlas. AHCY tissue expression.
- PMC11532973 — target-verified WB comparison
- PMC13538023 — target-verified WB comparison
- PMC9345734 — target-verified WB comparison