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- Table of Contents
NAB2 / NGFI-A-binding protein 2 · Western blot design guide
Design a Western Blot for NAB2
Source-linked NAB2 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NAB2 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
NAB2 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 | ~56.6 kDa | |
| Observed band | ~57 kDa | |
| Gel | 5–20% (catalog A02873-3) | |
| 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 + Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked NAB2 Western Blot Protocol Options
The A02873-3 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human MCF-7, human SiHa, rat thymus, mouse thymus (catalog A02873-3) |
| Gel % | 5–20% (catalog A02873-3) |
| Load | 30 ug; reducing conditions (catalog A02873-3) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A02873-3) |
| Membrane | nitrocellulose membrane (catalog A02873-3) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A02873-3) |
| Primary antibody | A02873-3 · 0.5 μg/mL (catalog A02873-3) |
| Primary incubation | overnight at 4°C (catalog A02873-3) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A02873-3) |
| Secondary incubation | 1.5 hour at RT (catalog A02873-3) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A02873-3) |
| Detection | ECL (catalog A02873-3) |
Section 2
What Is the Expected NAB2 Western Blot Band Size?
NAB2 is predicted at 56.6 kDa and observed near 57 kDa; the small difference has no established cause in the supplied evidence.
| Band near 57 kDa | Matches the empirical NAB2 band and its 56.6 kDa predicted mass; confirm identity with a specific control. |
| Several bands near the expected region | Could reflect isoforms 1, 2, and 3; their individual migration positions are unknown. |
| Doublet near 57 kDa | Could reflect NAB2 phosphorylation, although a visible shift is not established. |
| Stronger band in a nuclear fraction | Consistent with nuclear NAB2; isoform 2 is reported outside the nucleus. |
💡Expected NAB2 appearanceNAB2 has a predicted mass of 56.6 kDa and an empirical band near 57 kDa; confirm band identity with NAB2 depletion or another specific control.
| Predicted mass of 56.6 kDa | Provides the size estimate for the empirical band near 57 kDa. |
| Splice isoform 1 | May differ in size from isoforms 2 and 3; its individual mass is not supplied. |
| Splice isoform 2 | May differ in size from isoforms 1 and 3; its individual mass is not supplied. |
| Splice isoform 3 | May differ in size from isoforms 1 and 2; its individual mass is not supplied. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear NAB2 may be poorly recovered in the lysate. | Check a nuclear fraction and a positive control. |
| Band higher than expected | The higher band's identity is unestablished; NAB2 phosphorylation has no demonstrated shift here. | Compare with the 57 kDa band and test NAB2 depletion. |
| Band lower than expected | An isoform or degradation is possible; no lower NAB2 band size is established. | Use fresh lysate and test NAB2 depletion. |
| Multiple bands | NAB2 has three isoforms and documented phosphoserines, but their migration effects are unknown. | Test NAB2 depletion and, if needed, phosphatase treatment. |
| Weak or no signal | Nuclear NAB2 may be diluted in a whole-cell sample. | Check nuclear extraction and a positive control. |
| Fragments below expected size | Proteolysis or nonspecific antibody binding; no NAB2 fragment size is supplied. | Prepare fresh lysate with protease inhibitors and test NAB2 depletion. |
Experiment evidence
Western blot images showing NAB2 band
A02873-3 · NAB2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NAB2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NAB2 in Western blot, you can use lung lysate, which HPA rates High.
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: Lung and liver offer a High versus Not detected contrast, though isoform 2 is not nuclear.
HPA tissue expression evidence for NAB2
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 | macrophages | High | Protein (IHC) | HPA → |
| Placenta | decidual cells | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | glandular 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 → |
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Fallopian tube | glandular cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced NAB2 Western Blot Tips
Deeper troubleshooting and optimisation questions for NAB2, answered from its protein features.
How should NAB2 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 might NAB2 isoforms affect band interpretation?
Isoforms · UniProt lists three isoforms. Isoform 2 replaces residues 320–322 (LTI to ASP) and lacks residues 323–525; isoform 3 lacks residues 426–489. These sequence differences make smaller bands plausible. Check which isoforms the antibody can recognize before assigning a band.
Yes. Isoform 2 lacks canonical residues 323–525, and isoform 3 lacks residues 426–489. Either could contribute a smaller band if recognized by the antibody. Size alone cannot identify the isoform; check whether the antibody's target sequence is retained.
Yes. Isoform 2 lacks canonical residues 323–525, and isoform 3 lacks residues 426–489. Either could contribute a smaller band if recognized by the antibody. Size alone cannot identify the isoform; check whether the antibody's target sequence is retained.
Which NAB2 phosphorylation sites matter for Western blot design?
PTM · UniProt lists phosphoserines at canonical positions 6, 157, 159, 162, 171, and 479. Position 479 is absent from isoforms 2 and 3. State the UniProt coordinate convention when comparing these sites with antibody or paper numbering; the feature list alone does not establish a detectable band shift.
Does this guide establish induction of NAB2?
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 NAB2?
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 A02873-3 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.
What should be considered when quantifying NAB2 bands?
Quantitation · Decide whether the measurement covers all detectable isoforms or a specific band. Isoforms 2 and 3 have different sequences from canonical NAB2, and isoform 2 has different reported localization. Use the same band definition and sample fraction across comparisons.
Does the observed NAB2 band match its predicted mass?
Interpretation · The reported band at about 57 kDa closely matches the 56.6 kDa predicted mass of canonical NAB2. Its listed modifications do not, by themselves, establish a visible shift or explain any difference between calculated and apparent mass.
UniProt places NAB2 in the nucleus but notes that isoform 2 is not localized there. A nuclear fraction may therefore underrepresent isoform 2. Compare equivalent fractions when assessing isoform patterns.
UniProt places NAB2 in the nucleus but notes that isoform 2 is not localized there. A nuclear fraction may therefore underrepresent isoform 2. Compare equivalent fractions when assessing isoform patterns.
Boster reagents
NAB2 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-NAB2 Antibody Picoband®
Cat # A02873-3
The catalog reports one anti-NAB2 antibody, A02873-3, with reported human, mouse, and rat reactivity. Its Western blot image shows an approximately 57 kDa band in human cell and rat and mouse thymus lysates. No independent validation is supplied.
Which to pick: A02873-3 is the only listed option and has a Western blot image. The caption reports human MCF-7 and SiHa cells plus rat and mouse thymus, using 30 µg lysate per lane and 0.5 µg/mL primary antibody. These examples do not establish performance in every specimen.
Source: BosterBio NAB2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q15742.
- Human Protein Atlas. NAB2 tissue expression.
- PMC12393881 — target-verified WB comparison
- PMC5675621 — target-verified WB comparison
- PMC3804529 — target-verified WB comparison