Jurkat cells were treated for 30 min with 100 ug/ml ALLN (N-Acetyl-Leu-Leu-Norleucinal; a Calpain inhibitor and also proteasome inhibitor that prevents IkBa dephosphorylation) followed by incubation with (lanes 2 and 4) or without 1 nM TNF-a (1 and 3). The membranes were blotted with a IKB Alpha monoclonal antibody (Product # MA5-16152) (lanes 1 and 2) or a IKB Alpha monoclonal antibody (Product # MA5-16152) (that recognizes both non-phospho and phosphorylated forms of IkBa) and immunoreactivity was detected by ECL. The data shows that a IKB Alpha monoclonal antibody (Product # MA5-16152) detects specifically the phosphorylated form of IkBa.
|Tested species reactivity||Human, Mouse, Rat|
|Host / Isotype||Mouse / IgG1, kappa|
|Immunogen||A recombinant protein corresponding to amino acid residues 32-291 of human IkBa was used as immunogen.|
|Contains||0.05% sodium azide|
|Storage Conditions||Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||0.25-1 µg/10^6 cells|
|Immunohistochemistry (Paraffin) (IHC (P))||1:50|
|Western Blot (WB)||1-2 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Suggested positive control: Daudi, Jurkat, or NIH-3T3.
NF-kB is silenced in the cytoplasm by an inhibitory protein, IkB (1). Synthesis of IkBa is autoregulated (2). IkB proteins are phosphorylated by IkB kinase complex consisting of at least three proteins, IKK1/a, IKK2/b, and IKK3/g (3-6). External stimuli such as tumor necrosis factor or other cytokines results in phosphorylation and degradation of IkB releasing NF-kB dimers. NF-kB dimer subsequently translocates to the nucleus and activates target genes. Six members of IkB family members have been identified (1). One of the first gene induced following NF-kB activation is IkBa.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.