The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 36 kDa (predicted molecular weight: 36 kDa).
Use a concentration of 1 µg/ml.
Inhibits the activity of dimeric NF-kappa-B/REL complexes by trapping REL dimers in the cytoplasm through masking of their nuclear localization signals. On cellular stimulation by immune and proinflammatory responses, becomes phosphorylated promoting ubiquitination and degradation, enabling the dimeric RELA to translocate to the nucleus and activate transcription.
Involvement in disease
Ectodermal dysplasia, anhidrotic, with T-cell immunodeficiency autosomal dominant
Belongs to the NF-kappa-B inhibitor family. Contains 5 ANK repeats.
Phosphorylated; disables inhibition of NF-kappa-B DNA-binding activity. Phosphorylation at positions 32 and 36 is prerequisite to recognition by UBE2D3 leading to polyubiquitination and subsequent degradation. Sumoylated; sumoylation requires the presence of the nuclear import signal. Sumoylation blocks ubiquitination and proteasome-mediated degradation of the protein thereby increasing the protein stability. Monoubiquitinated at Lys-21 and/or Lys-22 by UBE2D3. Ubiquitin chain elongation is then performed by CDC34 in cooperation with the SCF(FBXW11) E3 ligase complex, building ubiquitin chains from the UBE2D3-primed NFKBIA-linked ubiquitin. The resulting polyubiquitination leads to protein degradation. Also ubiquitinated by SCF(BTRC) following stimulus-dependent phosphorylation at Ser-32 and Ser-36. Deubiquitinated by porcine reproductive and respiratory syndrome virus Nsp2 protein, which thereby interferes with NFKBIA degradation and impairs subsequent NF-kappa-B activation.
Cytoplasm. Nucleus. Shuttles between the nucleus and the cytoplasm by a nuclear localization signal (NLS) and a CRM1-dependent nuclear export.
Lane 1: Wild-type HAP1 whole cell lysate (20 µg) Lane 2: NFKBIA knockout HAP1 whole cell lysate (20 µg) Lane 3: HeLa whole cell lysate (20 µg) Lane 4: Jurkat whole cell lysate (20 µg) Lanes 1 - 4: Merged signal (red and green). Green - ab95338 observed at 38 kDa. Red - loading control, ab18058, observed at 130 kDa.
ab95338 was shown to specifically react with NFKBIA in wild-type HAP1 cells. No band was observed when NFKBIA knockout samples were examined. Wild-type and NFKBIA knockout samples were subjected to SDS-PAGE. Ab95338 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.
Western blot - IKB alpha antibody (ab95338)
All lanes : Anti-IKB alpha antibody (ab95338) at 1 µg/ml
Lane 1 : Human kidney tissue lysate - total protein (ab30203) Lane 2 : Human spleen tissue lysate - total protein (ab29699)
Lysates/proteins at 10 µg per lane.
Secondary Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 36 kDa Observed band size : 36 kDa
Exposure time : 4 minutes
Western blot - Anti-IKB alpha antibody (ab95338)
Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 36 kDa
Exposure time : 10 secondsAb95338 recognizes the tagged recombinant IKB alpha protein (ab59981) which has an expected molecular weight of 61kDa.
ICC/IF image of ab95338 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab95338, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.