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- Table of Contents
NFIX / Nuclear factor 1 X-type · Western blot design guide
Design a Western Blot for NFIX
Source-linked NFIX Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NFIX 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
NFIX 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 | ~55.1 kDa | |
| Observed band | ~55 kDa | |
| Gel | 5–20% (catalog A04138-1) | |
| Positive control | Breast (IHC candidate; verify WB) +4 more | |
| Negative control | Adrenal gland (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Methylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 6 isoform(s) |
Section 1
Source-Linked NFIX Western Blot Protocol Options
The A04138-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human A549, human U251, human PC-3, rat brain, mouse brain (catalog A04138-1) |
| Gel % | 5–20% (catalog A04138-1) |
| Load | 30 ug; reducing conditions (catalog A04138-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A04138-1) |
| Membrane | nitrocellulose membrane (catalog A04138-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A04138-1) |
| Primary antibody | A04138-1 · 0.5 μg/mL (catalog A04138-1) |
| Primary incubation | overnight at 4°C (catalog A04138-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A04138-1) |
| Secondary incubation | 1.5 hour at RT (catalog A04138-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A04138-1) |
| Detection | ECL (catalog A04138-1) |
Section 2
What Is the Expected NFIX Western Blot Band Size?
NFIX is predicted at 55.1 kDa and observed at ~55 kDa; no cause for the small difference is established.
| Band at ~55 kDa | Matches the empirical NFIX band and its 55.1 kDa predicted mass. |
| Additional bands near ~55 kDa | Could reflect NFIX isoforms or modified forms; their migration is not established. |
| Weak band in whole-cell lysate | Nuclear NFIX may be easier to detect after nuclear enrichment. |
| Higher band near ~110 kDa | Could represent retained NFIX homodimer, but a doubled Western-blot band is not established. |
💡Expected NFIX appearanceNFIX has a predicted mass of 55.1 kDa and an empirical band at ~55 kDa; confirm band identity with an NFIX depletion control, since its isoforms and modifications have no established migration pattern here.
| Predicted NFIX mass | 55.1 kDa predicts a band near the empirical ~55 kDa band. |
| Isoforms 1, 2, and 3 | Alternative splicing could change apparent size; individual masses are not supplied. |
| Isoforms 4, 5, and 6 | Alternative splicing could change apparent size; individual masses are not supplied. |
| DNA-binding homodimer | A retained dimer could appear higher, but its survival during Western-blot preparation is not established. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | NFIX is nuclear and may be dilute in whole-cell lysate. | Check nuclear enrichment and a positive lysate control. |
| Band higher than expected | NFIX forms homodimers, although a stable dimer band is not established. | Check reducing and denaturing conditions and confirm identity by NFIX depletion. |
| Band lower than expected | An alternative NFIX isoform is possible, but isoform masses are unavailable. | Confirm identity by NFIX depletion and check the antibody epitope. |
| Multiple bands | Six NFIX isoforms are annotated, without established distinct band positions. | Compare bands before and after NFIX depletion. |
| Weak or no signal | Nuclear NFIX may be underrepresented in the tested sample. | Check nuclear enrichment, sample loading, and a positive control. |
Experiment evidence
Western blot images showing NFIX band
A04138-1 · NFIX
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NFIX Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NFIX in Western blot, you can use breast tissue, where HPA reports high expression.
Positive control: Breast (IHC candidate; verify WB)
Negative control: Adrenal gland (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: NFIX is nuclear, so nuclear-enriched lysate may improve detection.
HPA tissue expression evidence for NFIX
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 |
|---|---|---|---|---|
| Breast | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Bergmann glia - nucleus | High | Protein (IHC) | HPA → |
| Cervix | squamous epithelial cells | High | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Pancreas | exocrine glandular cells | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Low | Protein (IHC) | HPA → |
| Endometrium | cells in endometrial stroma | Low | Protein (IHC) | HPA → |
Section 3
Advanced NFIX Western Blot Tips
Deeper troubleshooting and optimisation questions for NFIX, answered from its protein features.
How should NFIX 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 NFIX isoforms produce different bands?
Isoforms · Six isoforms are listed. Isoform 2 lacks residues 318–359; isoforms 3, 5, and 6 lack residues 442–502 and replace residues 419–441. These sequence differences could affect band size, but the features do not establish which isoforms are present in a sample.
Check the antibody epitope against the listed splice changes. Residues 318–359 are absent from isoform 2, while residues 442–502 are absent from isoforms 3, 5, and 6. Isoforms 4, 5, and 6 also have N-terminal changes. An epitope outside these regions offers broader potential coverage.
No. Its deletion of canonical residues 318–359 removes the regions containing phosphoserine 341 and asymmetric dimethylarginine 343. When comparing site-specific signals across isoforms, account for that deletion and check the numbering convention before assigning a band.
Check the antibody epitope against the listed splice changes. Residues 318–359 are absent from isoform 2, while residues 442–502 are absent from isoforms 3, 5, and 6. Isoforms 4, 5, and 6 also have N-terminal changes. An epitope outside these regions offers broader potential coverage.
No. Its deletion of canonical residues 318–359 removes the regions containing phosphoserine 341 and asymmetric dimethylarginine 343. When comparing site-specific signals across isoforms, account for that deletion and check the numbering convention before assigning a band.
Which annotated modifications should guide NFIX band interpretation?
PTM · UniProt lists phosphoserine at positions 265, 280, 288, 301, and 341, and asymmetric dimethylarginine at 343 and 390. These are UniProt coordinates; antibody or paper numbering may differ. Their presence alone does not prove a visible band shift.
The supplied features list no glycosylation sites or signal peptide. Glycosylation is therefore not a feature-supported explanation to prioritize for an unexpected NFIX band.
The supplied features list no glycosylation sites or signal peptide. Glycosylation is therefore not a feature-supported explanation to prioritize for an unexpected NFIX band.
Does this guide establish induction of NFIX?
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 NFIX?
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 A04138-1 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 NFIX Western blot signals be quantified?
Quantitation · Choose a measurement that matches the question: quantify the about 55 kDa band for the reported apparent NFIX signal, or measure resolved bands separately when assessing possible isoforms. An antibody against a variable region may miss some isoforms, so check its epitope before comparing totals.
Should NFIX run at its predicted mass?
Interpretation · Canonical NFIX is predicted at 55.1 kDa, close to the reported apparent band of about 55 kDa. The listed features do not establish a mass discrepancy or a visible shift.
Compare their sizes with the six listed isoforms and check whether the antibody recognizes the relevant sequence regions. NFIX also has annotated phosphorylation and methylation, but those annotations do not identify any extra band or prove a mobility shift. Confirm a band’s identity before assigning it to an isoform or modification.
Compare their sizes with the six listed isoforms and check whether the antibody recognizes the relevant sequence regions. NFIX also has annotated phosphorylation and methylation, but those annotations do not identify any extra band or prove a mobility shift. Confirm a band’s identity before assigning it to an isoform or modification.
Boster reagents
NFIX 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-NFIX Antibody Picoband®
Cat # A04138-1
the supplier A04138-1 is a rabbit polyclonal anti-NFIX antibody with a WB image showing an approximately 55 kDa band in human cell lines and rat and mouse brain lysates. The supplied evidence is one product image; no independent publication evidence is provided.
Which to pick: A04138-1 is the only listed option. Its stated reactivity is human, mouse, and rat, and its WB image includes human HeLa, A549, U251, and PC-3 cells plus rat and mouse brain lysates. Match your sample to these tested contexts.
Source: BosterBio NFIX gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q14938.
- Human Protein Atlas. NFIX tissue expression.
- PMC4793149 — target-verified WB comparison
- PMC12854730 — target-verified WB comparison
- PMC11144382 — target-verified WB comparison