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- Table of Contents
NFYA / Nuclear transcription factor Y subunit alpha · Western blot design guide
Design a Western Blot for NFYA
Source-linked NFYA Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NFYA 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
NFYA 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 | ~36.9 kDa | |
| Observed band | ~37 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Lung (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 | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked NFYA Western Blot Protocol Options
The M03524 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Target-positive lysate and matched negative control (standard starting point) |
| 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 | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | M03524 · 1:50 (catalog M03524) |
| 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 NFYA Western Blot Band Size?
NFYA's predicted mass is 36.9 kDa and its observed band is ~37 kDa; the supplied evidence does not establish a cause for their small difference.
| Band at ~37 kDa | Matches the observed NFYA band and its 36.9 kDa predicted mass |
| Two bands at different positions | Could reflect Long and Short isoforms if their migration differs; confirm band identity |
| One band despite two isoforms | The isoforms may not resolve as separate bands |
| Weak band in a cytosolic fraction | Consistent with NFYA's nuclear location |
💡Expected NFYA appearanceNFYA has a predicted mass of 36.9 kDa and an observed band at ~37 kDa; verify band identity with appropriate controls if additional bands appear.
| UniProt predicted mass | 36.9 kDa provides the sequence-based size reference; the observed band is ~37 kDa |
| Long isoform | May differ in size from Short; its individual migration is not supplied |
| Short isoform | May differ in size from Long; its individual migration is not supplied |
| Alternative splicing into Long and Short | Could produce different band positions if the isoforms resolve |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | NFYA may be below detection in the sample | Check loading and antibody performance with a positive-control lysate |
| Band higher than expected | The supplied features do not establish the band's identity or cause of higher migration | Compare with a positive control and verify antibody specificity |
| Band lower than expected | A Short isoform is possible, but its migration is not supplied | Check isoform expression and confirm band identity |
| Multiple bands | Long and Short isoforms could differ in migration | Compare isoform-specific controls and verify each band's identity |
| Weak or no signal | NFYA is nuclear and may be dilute in the tested fraction | Check a nuclear fraction alongside a positive control |
| Fragments below expected size | The supplied features do not identify an NFYA cleavage product | Check sample integrity and verify fragment identity with an independent antibody |
Experiment evidence
Western blot images showing NFYA band
M03524 · NFYA
M03524 · NFYA
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NFYA Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NFYA in Western blot, you can use lung tissue lysate, where HPA reports high expression.
Positive control: Lung (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: Adipose tissue is reported as not detected, providing a candidate negative control for this nuclear protein.
HPA tissue expression evidence for NFYA
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 |
|---|---|---|---|---|
| Lung | alveolar cells type I | High | Protein (IHC) | HPA → |
| Testis | spermatogonia cells | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | 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 → |
| Caudate | glial cells | Low | Protein (IHC) | HPA → |
| Hippocampus | glial cells | Low | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced NFYA Western Blot Tips
Deeper troubleshooting and optimisation questions for NFYA, answered from its protein features.
How should NFYA 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 should NFYA isoforms be quantified?
Isoforms · Decide whether to measure Long and Short separately or sum their signals, then use that choice consistently across samples. Short lacks canonical residues 26–54; an antibody targeting that segment could miss it.
Could phosphorylation shift the NFYA band?
PTM · UniProt lists phosphoserine at position 326 in canonical sequence numbering. This identifies a possible source of band heterogeneity, but the feature alone does not establish a visible shift. Compare matched samples with and without phosphatase treatment if phosphorylation is suspected.
Does this guide establish induction of NFYA?
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 NFYA?
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 M03524 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 NFYA be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Why does NFYA run near 37 kDa?
Interpretation · The observed band near 37 kDa agrees with the predicted 36.9 kDa mass. The supplied features do not establish a meaningful mass discrepancy or a visible shift.
NFYA has Long and Short isoforms. Short lacks residues 26–54 of the canonical sequence, so the forms may migrate differently. Whether they resolve as separate bands depends on the blot and antibody.
First compare bands with the expected approximately 37 kDa signal and consider the Long and Short isoforms. Phosphoserine 326 is documented, but neither that modification nor isoform presence proves the identity of an extra band. Confirm with an independent antibody or targeted validation.
UniProt places NFYA in the nucleus. A nuclear fraction is therefore a reasonable sample choice when whole-cell signal is weak. Compare equivalent fractions and loading across samples before interpreting signal changes.
NFYA associates with an NF-YB–NF-YC dimer to form a heterotrimeric transcription factor. That interaction alone does not identify a higher Western-blot band as the intact complex; assess the band with a method suited to testing protein association.
NFYA has Long and Short isoforms. Short lacks residues 26–54 of the canonical sequence, so the forms may migrate differently. Whether they resolve as separate bands depends on the blot and antibody.
First compare bands with the expected approximately 37 kDa signal and consider the Long and Short isoforms. Phosphoserine 326 is documented, but neither that modification nor isoform presence proves the identity of an extra band. Confirm with an independent antibody or targeted validation.
UniProt places NFYA in the nucleus. A nuclear fraction is therefore a reasonable sample choice when whole-cell signal is weak. Compare equivalent fractions and loading across samples before interpreting signal changes.
NFYA associates with an NF-YB–NF-YC dimer to form a heterotrimeric transcription factor. That interaction alone does not identify a higher Western-blot band as the intact complex; assess the band with a method suited to testing protein association.
Boster reagents
NFYA 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-NFYA Rabbit Monoclonal Antibody
Cat # M03524
M03524 is an anti-NFYA rabbit monoclonal antibody listed for human, mouse, and rat reactivity. Its supplied image is described as IP analysis with antibody at 1:50 and WB at 1:3,000; the excerpt does not identify the sample.
Which to pick: M03524 is the only listed NFYA antibody. It has a WB image, but the supplied caption describes IP analysis and gives no sample identity; check that your planned specimen and conditions fit before use.
Source: BosterBio NFYA gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P23511.
- Human Protein Atlas. NFYA tissue expression.
- PMC11456033 — target-verified WB comparison
- PMC13060261 — target-verified WB comparison
- PMC9452718 — target-verified WB comparison