IKK gamma Inhibitor Peptide Set

Functions as an IKK-Alpha/IKK-Beta decoy by binding to IKK-Gamma NBD, thereby preventing formation of the IKK complex.

The IKK-gamma inhibitory peptide contains a protein transduction (PTD) sequence (DRQIKIWFQNRRMKWKK) derived from antennapedia which renders the peptide cell permeable (Derossi et al, The third helix of the antennapedia homeodomain translocates through biological membranes. J Biol Chem. 269:10444-10450 (1994)] .The control peptide consists of only the PTD sequence.

1.IKK-gamma NEMO Binding Domain (NBD) Inhibitor Peptide: 2 x 1 mg (lyophilized) DRQIKIWFQNRRMKWKKTALDWSWLQTE (IKK-gamma/NEMO binding sequence is underlined). Molecular weight: 3692

2.Antennapedia Control peptide: 2 x 1 mg (lyophilized) DRQIKIWFQNRRMKWKK. Molecular weight: 2361

IKBKG

Inhibition of NF-kB activitiy in vivo and in vitro by interefering with IKK complex formation
Researchers can study the effect of NBD inhibitor peptide using a variety of methods. Quantitative readout assays include NF-kB/p65 ActivELISA Kit, Phospho-IkBalpha ActivELISA Kit and EMSA. Immunocytochemistry can also be used as a readout assay for visualizing the subcellular localization of NF-kB; activated NF-kB localizes to the nucleus, whereas NF-kB in the cytoplasm is generally considered be inactive.

U266 cells and EMSA assay are used to quality control every lot of the NBD inhibitor peptide set (Fig. 2). This protocol is written for U266, a human multiple myeloma cell line. Multiple myeloma is a B-cell malignancy, and a number of multiple myeloma cells lines, including U266, have been found to have constitutively active NF-kB. The EMSA assay shows that NBD suppressed the constitutive activation of NF-kB in U266 cells. The mmunocytochemistry data provides supporting evidence that the nuclear translocation of NF-kB was lost when the U266 cells were incubated with NBD (Fig.3).

Researchers must optimize assay methods for the NBD inhibitory peptide for different cell types. These include incubation time and amount of peptide used in an experiment. Depending on the cell types, morphology of cells may change after 2 hr of incubation with NBD peptide. For example, CHO cells become rounder in appearance after 2 hr incubation with NBD peptides. Since NF-kB is an important molecule for cell survival and proliferation, blockade of NF-kB activation by inhibiting IKK complex formation may prevent cell proliferation, which has been observed for CHO cells at 12 hrs. Researchers are advised to monitor the viability of cells for long-term incubation with the inhibitor.