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- Table of Contents
NFKB2 / Nuclear factor NF-kappa-B p100 subunit · Western blot design guide
Design a Western Blot for NFKB2
Real validated NFKB2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NFKB2 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
NFKB2 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 | ~96.7 kDa | |
| Observed band | ~110 kDa | |
| Gel | 5–20% (catalog M01228-1) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Cerebellum (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Ubl conjugation | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated NFKB2 Western Blot Protocols
The M01228-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Jurkat, human A549, human CACO-2 (catalog M01228-1) |
| Gel % | 5–20% (catalog M01228-1) |
| Load | 30 ug; reducing conditions (catalog M01228-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M01228-1) |
| Membrane | nitrocellulose membrane (catalog M01228-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M01228-1) |
| Primary antibody | M01228-1 · 1:1000 (catalog M01228-1) |
| Primary incubation | overnight at 4°C (catalog M01228-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:500 (catalog M01228-1) |
| Secondary incubation | 1.5 hour at RT (catalog M01228-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M01228-1) |
| Detection | ECL (catalog M01228-1) |
Section 2
What Is the Expected NFKB2 Western Blot Band Size?
NFKB2 p100 is predicted at 96.7 kDa, while an antibody QC blot shows approximately 110 kDa; the cause of this difference is not established.
| Band near 110 kDa | Empirical p100/p52 antibody band; its difference from the predicted mass is unexplained |
| Band near 96.7 kDa | Consistent with the predicted mass of full-length p100; confirm its identity |
| Band below full-length p100 | Could represent the p52 form; confirm with an antibody that recognizes the relevant region |
| Multiple bands | Could reflect p100, p52, or isoforms 1, 3, and 4; their separation is not established |
💡Expected NFKB2 appearanceFull-length NFKB2 has a predicted mass of 96.7 kDa, while an antibody QC blot shows a band at approximately 110 kDa; confirm band identity with appropriate controls because the cause of the difference is unknown.
| Predicted full-length p100 mass | 96.7 kDa is the sequence-based reference; the empirical band is approximately 110 kDa |
| Phosphorylation at Ser713, Ser715, and Ser717 | May affect migration, but no visible size change is established |
| Isoforms 1, 3, and 4 | May differ in size; individual masses and resolvable band positions are not supplied |
| p52 homodimer | Could appear higher if the complex survives electrophoresis; no dimer band size is established |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | The approximately 110 kDa empirical band exceeds the 96.7 kDa predicted mass for an undetermined reason | Compare with the antibody QC band and verify identity using NFKB2 depletion or an independent antibody |
| Band lower than expected | The antibody may detect the p52 form | Check antibody epitope coverage and compare with a p100-specific antibody |
| Multiple bands | p100, p52, or isoforms 1, 3, and 4 may contribute | Use epitope-specific antibodies and NFKB2 depletion to assign bands |
| Weak or no signal | NFKB2 occurs in both nucleus and cytoplasm, so recovery may vary with extraction | Check extraction of both compartments and confirm sample loading |
| Fragments below expected size | A smaller band could be p52 or sample degradation | Compare with a p52-reactive antibody and repeat preparation with protease inhibitors |
Experiment evidence
Western blot images showing NFKB2 band
M01228-1 · NFKB2
PB9150 · NFKB2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NFKB2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NFKB2 in Western blot, you can use appendix tissue lysate.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Cerebellum (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: NFKB2 occurs in both the nucleus and cytoplasm, so a single subcellular fraction may miss some signal.
HPA tissue expression evidence for NFKB2
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 |
|---|---|---|---|---|
| Appendix | lymphoid tissue | High | Protein (IHC) | HPA → |
| Lymph node | non-germinal center cells | High | Protein (IHC) | HPA → |
| Placenta | decidual cells | High | Protein (IHC) | HPA → |
| Skin | endothelial cells | High | Protein (IHC) | HPA → |
| Testis | spermatogonia cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Epididymis | glandular cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Ovary | follicle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NFKB2 Western Blot Tips
Deeper troubleshooting and optimisation questions for NFKB2, answered from its protein features.
How should NFKB2 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 NFKB2 isoforms affect band interpretation?
Isoforms · UniProt lists isoforms 1, 3 and 4. Isoform 3 replaces residues 374–428 and lacks residues 429–900, so its band could differ substantially from isoform 1. Isoform 4 lacks residue 860. These sequence differences alone do not establish where either isoform migrates.
An epitope within UniProt residues 429–900 is absent from isoform 3. Residues 374–428 are replaced in that isoform, so recognition there also depends on the antibody's exact epitope. Check the antibody's stated target region before comparing isoform bands.
An epitope within UniProt residues 429–900 is absent from isoform 3. Residues 374–428 are replaced in that isoform, so recognition there also depends on the antibody's exact epitope. Check the antibody's stated target region before comparing isoform bands.
Which phosphorylation sites matter when interpreting NFKB2 bands?
PTM · UniProt lists phosphoserine at 23, 161, 713, 715, 717, 812, 866 and 870, plus phosphothreonine at 429. These are UniProt coordinates; antibody or paper numbering may differ. Site presence alone does not establish a visible mobility shift.
UniProt attributes phosphorylation at serines 866 and 870 to MAP3K14. If comparing conditions that alter MAP3K14 activity, use an antibody whose target includes the relevant region and interpret any band change alongside an appropriate total NFKB2 measurement. The supplied features do not establish a specific shift or induction condition.
UniProt attributes phosphorylation at serines 866 and 870 to MAP3K14. If comparing conditions that alter MAP3K14 activity, use an antibody whose target includes the relevant region and interpret any band change alongside an appropriate total NFKB2 measurement. The supplied features do not establish a specific shift or induction condition.
Does this guide establish induction of NFKB2?
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 NFKB2?
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 M01228-1 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 cytoplasmic and nuclear NFKB2 bands be quantified?
Quantitation · UniProt places NFKB2 in both nucleus and cytoplasm and notes a cytoplasmic inactive form complexed with an inhibitor. Quantify fractions separately with consistent antibody detection and appropriate fraction controls; a change in one fraction alone need not represent a change in total NFKB2.
Why might NFKB2 appear near 110 kDa instead of 96.7 kDa?
Interpretation · The supplied observed band is approximately 110 kDa, while the predicted mass is 96.7 kDa. The listed phosphorylation sites and isoforms do not, by themselves, explain that difference. Treat 110 kDa as an empirical position and verify band identity with an appropriate control.
No. UniProt lists NFKB2 as a component of several p52-containing complexes, but the supplied features do not specify a p52 band mass or establish the identity of any smaller band. Consider the listed isoform sequences and the antibody epitope, then verify the band's identity with a suitable control.
No. UniProt lists NFKB2 as a component of several p52-containing complexes, but the supplied features do not specify a p52 band mass or establish the identity of any smaller band. Consider the listed isoform sequences and the antibody epitope, then verify the band's identity with a suitable control.
Boster reagents
NFKB2 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-NFkB p100 / p52 Rabbit Monoclonal Antibody
Cat # M01228-1
Real WB data
Anti-NFkB/NFKB2 p100/p52 Antibody Picoband®
Cat # PB9150
Two the supplier anti-NFKB2 antibodies have WB images. M01228-1 shows an approximately 110 kDa band in human, rat, and mouse samples; PB9150 shows approximately 52 and 120 kDa bands in human and mouse samples. These images document the listed conditions, not broader validation.
Which to pick: Choose M01228-1 for a documented rat sample or an approximately 110 kDa band. Choose PB9150 if you need an image showing both the 52 kDa active form and 120 kDa precursor. Both list human, mouse, and rat reactivity; PB9150’s image lacks rat samples.
Source: BosterBio NFKB2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q00653.
- Human Protein Atlas. NFKB2 tissue expression.
- PMC13367193 — target-verified WB comparison
- PMC5772637 — target-verified WB comparison
- PMC10730750 — target-verified WB comparison