NF-KB p105 Mouse mAb: Selection & Application Guide

NF-KB p105 Mouse mAb is a monoclonal antibody specifically raised against the NF-κB p105 precursor protein, designed to support reliable detection and functional analysis of this critical transcription factor subunit across a range of immunological assay formats.

Product SKU

Ab349141

Antibody Format

Mouse Monoclonal Antibody (mAb)

Target Protein

NF-κB p105 (NFKB1 gene product; precursor of p50)

Recommended Applications

Western Blot (WB), Immunohistochemistry (IHC), Immunoprecipitation (IP)

Biological Significance

NF-κB p105 is both an NF-κB subunit precursor and an IκB-like inhibitor; central to inflammatory and immune signaling

Host Species

Mouse

Background: NF-κB p105 in Cell Signaling

NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) is a family of transcription factors that regulate genes involved in inflammation, immunity, cell survival, and proliferation. The p105 protein, encoded by the NFKB1 gene, is unique among NF-κB family members in that it serves a dual role: it is the full-length precursor of the p50 subunit, generated by constitutive or signal-induced proteasomal processing, and it independently functions as an IκB-like inhibitor that retains NF-κB complexes in the cytoplasm through its C-terminal ankyrin repeat domain.

Upon appropriate stimulation—such as by cytokines (TNF-α, IL-1β), lipopolysaccharide (LPS), or antigen receptor engagement—p105 undergoes signal-induced degradation or processing, liberating p50-containing dimers that translocate to the nucleus to activate target gene transcription. The p105/p50 axis is therefore a critical regulatory node in both canonical and non-canonical NF-κB signaling, making antibodies against p105 indispensable tools for dissecting pathway activation states.

Antibody Specifications and Validation

The NF-KB p105 Mouse mAb (SKU: Ab349141) is a murine-derived monoclonal antibody offering high specificity and lot-to-lot consistency compared with polyclonal alternatives. Key specification considerations include:

• Isotype: Confirm the IgG isotype with your received datasheet to select an appropriate secondary antibody.
• Immunogen: Typically directed against a specific epitope within the p105 sequence; epitope location (N-terminal, central, or C-terminal) determines whether the antibody recognizes both p105 and processed p50, or p105 exclusively. Refer to the product datasheet for epitope details.
• Specificity: Validated by observing a band at the expected molecular weight (~105 kDa for p105; ~50 kDa for p50 if the epitope is within the shared N-terminal region) in western blot using appropriate positive control lysates (e.g., Jurkat, HeLa, or RAW264.7 cells stimulated with LPS or TNF-α).
• Cross-reactivity: Consult the datasheet for confirmed species cross-reactivity (human, mouse, rat). Monoclonal antibodies may have narrower cross-reactivity than polyclonals.

All Aladdin Scientific antibodies referenced here correspond to supplier catalog entry absea#KC-18067. Researchers should always verify specifications against the current product datasheet prior to experimental use.

Recommended Protocols by Application

The following application-specific guidance applies to Ab349141. Always confirm optimal dilutions by titration using your specific cell or tissue lysates.

Western Blot (WB)

1. Lyse cells in RIPA buffer supplemented with protease and phosphatase inhibitors. For NF-κB pathway studies, include phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride) to preserve phosphorylation states.
2. Resolve 20–40 µg total protein on a 7.5–10% SDS-PAGE gel (p105 at ~105 kDa; p50 at ~50 kDa).
3. Transfer to PVDF membrane and block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.
4. Incubate with primary antibody at the manufacturer-recommended dilution overnight at 4 °C.
5. Wash 3× with TBST, incubate with HRP-conjugated anti-mouse secondary antibody, and detect by ECL.
6. Positive control lysates: LPS-stimulated RAW264.7 macrophages or TNF-α-stimulated HeLa cells.

Immunohistochemistry (IHC)

1. Fix tissue in 10% neutral-buffered formalin and embed in paraffin (FFPE). Antigen retrieval by heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) is typically required for nuclear/cytoplasmic targets.
2. Block endogenous peroxidase with 3% H₂O₂ for 10 minutes. Block non-specific binding with 5–10% normal serum from the secondary antibody host species.
3. Apply primary antibody at recommended dilution; incubate 1 hour at room temperature or overnight at 4 °C in a humidified chamber.
4. Detect with species-appropriate HRP-polymer secondary system and DAB chromogen; counterstain with hematoxylin.

Immunoprecipitation (IP)

1. Lyse cells in mild lysis buffer (e.g., 0.5% NP-40 in PBS with inhibitors) to preserve protein–protein interactions.
2. Pre-clear lysate with Protein A/G agarose beads for 1 hour at 4 °C.
3. Add Ab349141 at 1–5 µg per 500 µg total protein; rotate overnight at 4 °C.
4. Capture immune complexes with Protein A/G beads, wash 3–5× with lysis buffer, elute in SDS sample buffer, and analyze by WB.

Storage, Handling, and Quality Control

Proper storage and handling are essential to maintain antibody activity and ensure reproducible results:

• Storage temperature: Store at –20 °C for long-term storage. Avoid repeated freeze–thaw cycles; aliquot upon receipt if multiple uses are anticipated.
• Short-term storage: For working stocks used within 1–2 weeks, antibodies may be stored at 4 °C. Do not store at 4 °C for extended periods as this may lead to aggregation and loss of activity.
• Carrier protein: If the formulation contains BSA or glycerol, ensure compatibility with your downstream application (BSA may interfere with certain proximity-ligation or mass spectrometry assays).
• Sterility: Maintain aseptic technique when aliquoting. Avoid sodium azide-containing formulations if the antibody is to be used in cell-based functional assays, as azide is cytotoxic.
• Lot-to-lot consistency: Always record the lot number. If switching lots, perform a side-by-side validation western blot before committing to large experimental sets.

For quality control and regulatory compliance purposes, retain the Certificate of Analysis (CoA) associated with each lot used in your experiments.

Troubleshooting Common Issues

The table below summarizes common problems encountered when using anti-p105 monoclonal antibodies and recommended corrective actions:

Problem	Likely Cause	Recommended Action
No band detected by WB	Insufficient antigen, incorrect molecular weight range, or antibody degradation	Confirm positive control lysate expresses p105 (use stimulated immune cells); verify gel percentage (7.5–10%); check antibody storage history
Multiple non-specific bands	Antibody concentration too high or insufficient blocking	Titrate antibody concentration downward; increase blocking time or switch to BSA block; add 0.1% Tween-20 to wash buffer
High background in IHC	Insufficient endogenous peroxidase quenching or non-specific secondary binding	Extend H₂O₂ quenching step; optimize serum blocking; include negative control (no primary antibody) on each run
Weak or absent IP pulldown	Epitope masked in native conditions or insufficient antibody	Optimize lysis buffer stringency; increase antibody amount; confirm target expression by direct WB before IP

Frequently asked questions

Can Ab349141 detect both p105 and p50 by western blot?

Whether Ab349141 detects p50 (the proteolytically processed form of p105, ~50 kDa) depends on the location of its immunizing epitope. If the epitope resides within the N-terminal region shared by both p105 and p50, both bands may be visible. If the epitope is within the C-terminal ankyrin repeat domain unique to p105, only the ~105 kDa band will appear. Consult the product datasheet for confirmed epitope information.

Which cell lines serve as appropriate positive controls for NF-κB p105 detection?

Cell lines with high NF-κB pathway activity are ideal positive controls. Jurkat (human T-cell leukemia), HeLa (human cervical carcinoma), and RAW264.7 (mouse macrophage) cells all express endogenous NF-κB p105 and can be further stimulated with TNF-α (10–20 ng/mL) or LPS (1 µg/mL) to enhance p105 expression and observe processing to p50. Always include an unstimulated control lane for comparison.

Is Ab349141 suitable for use in flow cytometry or ELISA?

The product source material does not confirm validation of Ab349141 for flow cytometry or ELISA. Applying an antibody in an application for which it has not been validated can produce unreliable results. Contact Aladdin Scientific technical support to inquire about validated applications and to request application-specific validation data before proceeding with untested formats.

How should I validate a new lot of Ab349141 before starting a large experiment?

Run a side-by-side western blot comparing the new lot with your previous validated lot using the same positive control lysate and antibody dilution. Confirm that band intensity, molecular weight, and specificity profile are equivalent. Document the lot number, dilution used, and results in your laboratory notebook as part of your quality control record.

What is the significance of p105 phosphorylation in NF-κB signaling?

Phosphorylation of p105, primarily at serine residues within its PEST domain (e.g., Ser927 and Ser932 in the human sequence), by IKKβ upon stimulation targets p105 for ubiquitination and proteasomal degradation or processing, thereby releasing p50-containing dimers and free NF-κB subunits. Using phospho-specific antibodies alongside total p105 antibodies like Ab349141 allows researchers to distinguish between total protein levels and activation-associated processing events.

References

1. Hayden MS, Ghosh S. NF-κB, the first quarter-century: remarkable progress and outstanding questions. Genes Dev. 2012;26(3):203-234. doi:10.1101/gad.183434.111
2. Moorthy AK, Bhatt DL, Bhatt D, et al. IκB kinase-induced p105 processing controls NF-κB activation and signaling specificity. EMBO J. 2006;25(21):4975-4985.
3. Aladdin Scientific. Product Datasheet: NF-KB p105 Mouse mAb, Ab349141 (absea#KC-18067). Aladdin Scientific; [date on file].
4. Ghosh S, Hayden MS. New regulators of NF-κB in inflammation. Nat Rev Immunol. 2008;8(11):837-848. doi:10.1038/nri2423

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.