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FANCD2 / Fanconi anemia group D2 protein · Western blot design guide
Design a Western Blot for FANCD2
Real validated FANCD2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-FANCD2 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
FANCD2 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 | ~164.1 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Testis (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 + Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 4 isoform(s) |
Section 1
Real Curated FANCD2 Western Blot Protocols
The A00563 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | various cells (catalog A00563) |
| Gel % | 8–10% (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 | A00563 · 1:500 (catalog A00563) |
| 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 FANCD2 Western Blot Band Size?
FANCD2 has a predicted 164.1 kDa monomer; isoforms and homodimerization could affect patterns, but no empirical migration is supplied.
| Band near 164.1 kDa | Consistent with the predicted FANCD2 monomer; confirm identity with appropriate controls |
| Band near twice the monomer size | Could reflect a homodimer if it survives electrophoresis; no inter-chain disulfide is listed |
| Several bands at different positions | Could reflect FANCD2 isoforms 1, 2, 3, and 4; distinct migration is unverified |
| Weak band in a cytoplasmic fraction | FANCD2 is nuclear, so nuclear enrichment may improve detection |
💡Expected FANCD2 appearanceUniProt predicts a 164.1 kDa FANCD2 monomer; no empirical band size is supplied, and the listed isoforms and homodimer do not establish additional blot positions without band-identity controls.
| Predicted monomer mass | Places full-length FANCD2 near 164.1 kDa by sequence |
| Isoform 1 | May differ in size from other named isoforms; its mass is not supplied |
| Isoform 2 | May differ in size from other named isoforms; its mass is not supplied |
| Isoforms 3 and 4 | May differ in size from other named isoforms; their masses are not supplied |
| Homodimer | Could appear near twice the monomer size if the complex survives electrophoresis |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear FANCD2 may be underrepresented in the sampled fraction | Check a nuclear extract and a nuclear fraction marker |
| Band higher than expected | A FANCD2 homodimer may persist, though its migration is unverified | Compare denaturing conditions and confirm the band with FANCD2 depletion |
| Band lower than expected | An alternative isoform or protein breakdown is possible | Compare antibodies targeting different regions and confirm with FANCD2 depletion |
| Multiple bands | Four named isoforms are listed, but their band positions are unknown | Check which bands decrease after FANCD2 depletion |
| Weak or no signal | The nuclear target may be poorly recovered | Verify nuclear extraction and sample loading |
| Fragments below expected size | Protein breakdown during preparation is possible | Prepare fresh lysate with protease inhibitors and compare antibody epitopes |
Experiment evidence
Western blot images showing FANCD2 band
A00563 · FANCD2
M00563 · FANCD2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for FANCD2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for FANCD2 in Western blot, you can use testis lysate, as HPA reports high expression in testis.
Positive control: Testis (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 the samples.
⚠️Feasibility: HPA provides a not-detected tissue control, and FANCD2’s nuclear location may favor nuclear-enriched lysate for detection.
HPA tissue expression evidence for FANCD2
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 |
|---|---|---|---|---|
| Testis | pachytene spermatocytes | High | Protein (IHC) | HPA → |
| Colon | glandular cells | Medium | Protein (IHC) | HPA → |
| Duodenum | glandular cells | Medium | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Nasopharynx | ciliated cells (cell body) | Medium | 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | basal cells | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced FANCD2 Western Blot Tips
Deeper troubleshooting and optimisation questions for FANCD2, answered from its protein features.
How should FANCD2 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.
How could FANCD2 isoforms affect band interpretation?
Isoforms · Four isoforms are listed. Isoform 4 changes residues 232–241 and lacks 242–1451; isoform 3 changes 1229–1249 and lacks 1250–1451; isoform 1 replaces 1428–1451. These sequence differences could affect antibody recognition and band size. Check the antibody epitope before assigning a band to an isoform.
Which FANCD2 phosphorylation sites matter when interpreting bands?
PTM · UniProt lists phosphoserine at 8, 222, 592, 594, 717, 1257, 1401, 1404, 1412, 1423 and 1435, plus phosphothreonine at 1426. ATM is specified for serines 222, 1401 and 1404. These are UniProt coordinates; paper or antibody numbering may differ. The site annotations do not establish a visible band shift.
When should FANCD2 induction be assessed?
Induction · FANCD2 concentrates in nuclear foci during S phase and upon genotoxic stress, according to the supplied localization note. Compare matched cell cycle or stress conditions when assessing its localization or abundance. Nuclear foci indicate redistribution and do not, by themselves, establish increased total protein.
What transfer method to use for FANCD2 Western blot?
Transfer · FANCD2 has a predicted mass of 164.1 kDa. Choose a transfer protocol validated for proteins around that size and check transfer with a membrane stain. The supplied features do not specify a membrane, transfer apparatus or settings.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A00563 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 FANCD2 bands be quantified across conditions?
Quantitation · Use the same band definition and normalization approach across samples, and record cell cycle and genotoxic stress conditions. FANCD2 is nuclear and concentrates in foci during S phase and stress, so differences in sample preparation or nuclear recovery could affect measured signal. Verify that the quantified band corresponds to the intended isoform.
Why might FANCD2 migrate differently from its predicted mass?
Interpretation · The supplied sequence has a predicted mass of 164.1 kDa, but no observed band size is available. Phosphorylation and alternative isoforms are annotated; their presence alone does not establish a visible shift or explain a mass difference. Compare the observed band with a molecular weight marker and check which isoform the antibody recognizes.
Consider the annotated shorter isoforms 3 and 4, especially if the antibody recognizes a region they retain. Check the antibody epitope against their deleted regions. Phosphorylation is annotated, but does not alone identify an extra band or prove a mobility shift. No empirical FANCD2 band size was supplied.
Consider the annotated shorter isoforms 3 and 4, especially if the antibody recognizes a region they retain. Check the antibody epitope against their deleted regions. Phosphorylation is annotated, but does not alone identify an extra band or prove a mobility shift. No empirical FANCD2 band size was supplied.
Boster reagents
FANCD2 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-FANCD2 Antibody
Cat # A00563
Real WB data
Anti-FANCD2 Rabbit Monoclonal Antibody
Cat # M00563
A00563 and M00563 are anti-FANCD2 antibodies with WB images and listed human, mouse, and rat reactivity. M00563 shows an approximately 164 kDa band in human HeLa, HepG2, and MCF-7 lysates. The supplied captions do not document mouse or rat WB samples.
Which to pick: For human whole-cell lysates, M00563 has a documented 1:1000 WB example. A00563 shows WB on nuclear extracts from unspecified cells at 1:500. Both have WB images; choose based on your sample preparation and confirm performance in your own samples.
Source: BosterBio FANCD2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9BXW9.
- Human Protein Atlas. FANCD2 tissue expression.
- PMC9369391 — target-verified WB comparison
- PMC3509204 — target-verified WB comparison
- PMC3048478 — target-verified WB comparison