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- Table of Contents
AFP · Western blot design guide
AFP Western Blot Planning Guide
Plan a AFP Western blot around the catalog-observed 68.7 kDa band, image-backed A00522-3 evidence, HPA controls, and verified protocol records.
Evidence assembled July 2026 · For research use; verify linked source records and product datasheet before use
AFP 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 | 68.7 kDa observed | |
| Observed band | 68.7 kDa observed | |
| Gel | 12-15% | |
| Positive control | Adrenal gland | |
| Negative control | Adrenal gland |
Important caveats
Reasons your observed band may differ from the expected size.
| ⓘ | Calculated mass | 68.7 kDa |
| ⓘ | Localization | Secreted |
| ⓘ | Processing / PTM | Record-dependent |
| ⓘ | Reactivity | Human |
Section 1
Real Curated AFP Western Blot Protocols
Start with the molecular-weight rule, then compare verified publication-derived conditions.
| Sample / lysate | Adipose tissue |
| Gel % | 12-15% |
| Load | 20-30 µg total protein per lane |
| Transfer | Semi-dry, standard transfer |
| Membrane | 0.45 µm PVDF |
| Blocking | 5% non-fat milk or 5% BSA in TBST |
| Primary | A00522-3 at datasheet starting dilution |
| Primary incubation | Overnight at 4 °C with gentle agitation |
| Secondary | Species-matched HRP conjugate at validated dilution |
| Wash | 3 × 5 min in TBST |
| Detection | Chemiluminescent substrate |
| Exposure | Bracket exposures to avoid saturation |
Section 2
What Is the Expected AFP Western Blot Band Size?
Use the product-observed 68.7 kDa band as the primary planning value and retain the UniProt calculated mass as context.
| 68.7 kDa | Matches the authoritative product WB observation. |
| 68.7 kDa calculated | Use as UniProt context, not as a replacement observed band. |
| Unexpected additional signal | Do not assign identity without orthogonal positive/negative controls. |
💡Expected AFP appearancePlan around 68.7 kDa and keep the calculated mass as supporting context.
| Catalog-observed band | 68.7 kDa; use this as the primary experimental expectation. |
| Calculated mass | 68.7 kDa from UniProt P02771; retain as context. |
| Gel selection | 12-15%; shared with the recommended protocol and poster. |
| Specificity check | Compare the lead HPA positive and negative controls with A00522-3. |
| Situation | Likely cause | Next action |
|---|---|---|
| 68.7 kDa | Matches the authoritative product WB observation. | Confirm with orthogonal controls and the linked product record. |
| Additional band | May reflect processing, modification, or non-specific signal. | Run a dilution series and compare positive/negative controls. |
| Weak signal | Target abundance or transfer may be limiting. | Verify transfer, increase positive-control abundance, and bracket exposure. |
Experiment evidence
Western blot images showing AFP band
A00522-3 · AFPObserved 68.7 kDa · product-specific WB image
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for AFP Western blot
🧪Use Adrenal gland as the first positive-control candidate and retain an orthogonal negative control.
Positive control: Adrenal gland (Not detected)
Negative control: Adrenal gland (Not detected)
HPA protein score determines control status; other expression data is supporting context only.
HPA tissue expression evidence for AFP
Comprehensive Human Protein Atlas IHC scoring per tissue. Rows are taken directly from the HPA tissue chart on each tissue page — 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
The Human Protein Atlas exposes no structured IHC tissue profile for AFP, so tissue-level positive/negative controls cannot be listed here. Use a documented AFP-positive lysate (recombinant AFP or a known-expressing line) as the positive control and a AFP knockdown/knockout lysate as the negative control; confirm against the antibody datasheet.
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Reported tissue cells | Not detected | Protein (HPA) | HPA → |
| Adipose tissue | Reported tissue cells | Not detected | Protein (HPA) | HPA → |
| Appendix | Reported tissue cells | Not detected | Protein (HPA) | HPA → |
| Bone marrow | Reported tissue cells | Not detected | Protein (HPA) | HPA → |
| Adipose tissue | Reported tissue cells | Not detected | Protein (HPA) | HPA → |
| Appendix | Reported tissue cells | Not detected | Protein (HPA) | HPA → |
Section 3
Advanced AFP Western Blot Tips
Deeper troubleshooting and optimisation questions for AFP, answered from its protein features.
Which band should guide the blot?
Use 68.7 kDa, the observation attached to the authoritative A00522-3 WB record.
How should calculated mass be interpreted?
Treat the UniProt calculated mass as context; it does not replace the catalog-observed 68.7 kDa expectation.
Which positive control should I start with?
Start with Adrenal gland, the lead HPA protein-expression candidate.
Which negative control is defensible?
Use Adrenal gland as an orthogonal HPA Not detected candidate.
Which gel should I use?
Use 12-15% consistently across the quick facts, protocol table, and poster.
What transfer method to use for AFP Western blot?
Use the transfer method in the recommended protocol and verify transfer before blocking.
How should A00522-3 be started?
Start at the linked datasheet condition and run a three-point primary-antibody dilution test.
Which publication-derived protocols can I compare?
No verified publication protocol was supplied; use only the deterministic recommended protocol.
Boster reagents
AFP Western Blot Reagents
Human-reactive AFP Western blot reagents with authoritative product imagery.
Real WB data
Anti-alpha 1 Fetoprotein/AFP Antibody Picoband®
Cat # A00522-3
Real WB data
Anti-alpha 1 Fetoprotein/AFP Antibody Picoband®
Cat # PB9090
Only image-backed, WB-validated Human/Mouse/Rat recommendations from the prepared catalog evidence are shown.
Source: prepared picoband-wb product evidence; each card retains its own SKU, URL, observed band, and authoritative WB image.