Western blot shows lysates of Nalm‑6 human Pre‑B acute lymphocytic leukemia cell line. PVDF membrane was probed with 1 µg/mL of Goat Anti-Human Ikaros Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4984) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF019). Bands were detected for Ikaros (1-8 spice forms) at approximately 37 to 63 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 8.
Detection of Ikaros-regulated Genes by Chromatin Immunoprecipitation.
Jurkat human acute T cell leukemia cell line treated with 50 ng/mL PMA and 200 ng/mL calcium ionomycin for 30 minutes was fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. Ikaros/DNA complexes were immunoprecipitated using 5 μg Goat Anti-Human Ikaros Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4984) or control antibody (Catalog # AB-108-C) for 15 minutes in an ultrasonic bath, followed by Biotinylated Anti-Goat IgG Secondary Antibody (Catalog # BAF109). Immunocomplexes were captured using 50 μL of MagCellect Streptavidin Ferrofluid (Catalog # MAG999) and DNA was purified using chelating resin solution. The VPAC promoter was detected by standard PCR.
Detection of Ikaros in Jurkat Human Cell Line by Flow Cytometry.
Jurkat human acute T cell leukemia cell line was stained with Goat Anti-Human Ikaros Antigen Affinity‑purified Polyclonal Antibody (Catalog # AF4984, filled histogram) or control antibody (Catalog # AB‑108‑C, open histogram), followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with methanol.
Detection of Human Ikaros by Simple WesternTM.
Simple Western lane view shows lysates of Nalm‑6 human Pre-B acute lymphocytic leukemia cell line, loaded at 0.2 mg/mL. Specific bands were detected for Ikaros at approximately 63-77 kDa (as indicated) using 10 µg/mL of Goat Anti-Human Ikaros Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4984) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.
Preparation and Storage
Reconstitute at 0.2 mg/mL in sterile PBS.
Reconstitution Buffer Available
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. *Small pack size (SP) is shipped with polar packs. Upon receipt, store it immediately at -20 to -70 °C
Stability & Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
12 months from date of receipt, -20 to -70 °C as supplied.
1 month, 2 to 8 °C under sterile conditions after reconstitution.
6 months, -20 to -70 °C under sterile conditions after reconstitution.
Ikaros (also LyF1) is a 60 kDa member of the C2H2-type zinc-finger protein family. It is found in both T and B cells and serves as a context‑dependent activator or repressor of genes. Human Ikaros is 519 amino acids (aa) in length. It possesses two zinc‑finger domains, one at the N‑terminus that contains four zinc-finger motifs (aa 117‑224) and one at the C‑terminus that contains two zinc‑finger motifs (aa 462‑514). The C‑terminal motifs mediate homo- or heterodimerization, while the N‑terminal motifs bind DNA. Multiple splice forms of 37 kDa to 47 kDa exist (Ikaros 2‑8) with reduced numbers of N‑terminal zinc-finger motifs. At least three are needed for DNA binding, and a heterodimer with a two‑motif monomer is suggested to be transcriptionally inert. Over aa 427‑519 (which are not spliced), human Ikaros shows 88% aa identity to mouse Ikaros.
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