NF-κB p65 (RELA)

Structure of the NF-κB p65 target protein

Figure 1: Structure of the NF-κB p65 target protein.

Introduction to NF-κB p65

Protein Function

NF-kappa-B is a pleiotropic transcription factor which is present in almost all cell types and is involved in many biological processed such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis.
In mammals, the NF-κB family consists of five related transcription factors containing Rel-like domains: P50, P52, RelA (p65), c-Rel, and RelB.
NF-κB is regulated by various mechanisms including post-translational modifications, subcellular compartmentalization, and interactions with cofactors or corepressors.
NF-kappa-B complexes are held in the cytoplasm in an inactive state complexed with members of the NF-kappa-B inhibitor (I-kappa-B) family. In a conventional activation pathway, I-kappa-B is phosphorylated by I-kappa-B kinases (IKKs) in response to different activators, subsequently degraded thus liberating the active NF-kappa-B complex which translocates to the nucleus and regulate gene expression through transcription factors.
Post-translational modifications, such as phosphorylation and acetylation of p65-NF-κB subunit, have recently been found to regulate NF-κB transcriptional activity

Protein Characteristics

NF-κB p65 undergoes various post-translational modifications including phosphorylation, ubiquitination, and acetylation.
Post-translational modifications of NF-κB p65 may be conditionally induced; for example, phosphorylation on Ser-536 of NF-κB p65 protein can be detected after cell stimulation with Calyculin A and TNF-α.
Due to these post-translational modifications, NF-κB p65 protein band size and numbers may be affected in Western blot.

Protein Localization

NF-κB p65 is localized in both the nucleus and cytoplasm, predominantly in the nucleus under normal conditions, but also found in the cytoplasm in an inactive form.

ICC Experimental Results of NF-κB p65 Protein, Anti-NF-κB p65 Antibody (ab76311). Green: NF-κB p65, Blue: DAPI

Figure 2: ICC Experimental Results of NF-κB p65 Protein, Anti-NF-κB p65 Antibody (ab76311). Green: NF-κB p65, Blue: DAPI.

Isoforms & Post-translational Modifications

Human (Q04206): Isoforms 1-4: 59-60 kDa (predicted)
Mouse (Q04207): Isoforms p65 and p65 delta: 59-60 kDa (predicted)
Rat (Q7TQN4): 60 kDa (predicted)
Phosphorylation
Ubiquitination
Methylation
Acetylation

WB Experiment Tips

Precautions:

Multiple bands may appear in Western blot experiments due to the various post-translational modifications of NF-κB p65.
Detected band sizes in Western blot experiments may differ from predicted sizes due to the multiple post-translational modifications of NF-κB p65.
Inducing conditions may be necessary for detecting NF-κB p65 post-translational modifications; thus, induction conditions can help address weak or absent signals.
Use freshly prepared samples to prevent weakening of phosphorylation modifications in frozen samples. When detecting phosphorylation modifications, confirm the total NF-κB p65 protein content in the cells first.
NF-κB p65 protein has multiple potential phosphorylation modification sites. Refer to literature to identify specific sites of phosphorylation induced by stimulation and choose appropriate antibodies. Setting up positive controls is recommended.

Positive Controls

NF-κB p65: HeLa whole cell lysate (ab150035).
NF-κB p65 (phospho S536): HeLa whole cell lysate treated with Calyculin A and TNF-alpha.

Example Results

WB with Anti-NF-κB p65 (phospho S536) Antibody (ab76302)

Figure 3: WB with Anti-NF-κB p65 (phospho S536) Antibody (ab76302)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) treated with Calyculin A and TNF-alpha whole cell lysate

Predicted band size: 60 kDa
Detected band size: 65 kDa

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample preparation:

Add a protease inhibitor cocktail to prevent degradation of target proteins.
For phosphorylated proteins, add enough phosphatase inhibitor cocktail to prevent dephosphorylation during extraction.
For acetylated modified proteins, add a enough deacetylase inhibitor cocktail to prevent deacetylation of the target protein.
Keep samples on ice throughout the entire sample preparation process.
Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

Load at least 20μg total protein for electrophoresis.

Transfer:

It is recommended to stain the membrane with Ponceau S after the transfer to confirm the success of the transfer.
It is recommended not to cut the membrane and keep the entire membrane or at least a portion of the membrane (50-100kDa) for antibody incubation.