Whole cell extracts (190 µg total protein) prepared from Jurkat cells were immunoprecipitated overnight with 2.5 µg of purified Armenian Hamster IgG isotype ctrl antibody (clone HTK888) (Cat. No. 400902) or purified anti-mouse/human Helios antibody (clone 22F6) (Cat. No. 137202). The resulting IP fractions and whole cell extract input (10%) were resolved by 4-12% Bis-Tris gel electrophoresis, transferred to a PVDF membrane and probed with a rabbit antibody against human Helios overnight at 4°C. Proteins were visualized by chemiluminescence detection using HRP donkey anti-rabbit IgG (minimal x-reactivity) antibody (Cat. No. 406401) at a 1:3000 dilution. Lane M: Molecular weight marker.
Whole cell extracts (190 µg total protein) prepared from Jurkat cells were immunoprecipitated overnight with 2.5 µg of purified Armenian Hamster IgG isotype ctrl antibody (clone HTK888) (Cat. No. 400902) or purified anti-mouse/human Helios antibody (clone 22F6) (Cat. No. 137202). The resulting IP fractions and whole cell extract input (10%) were resolved by 4-12% Bis-Tris gel electrophoresis, transferred to a PVDF membrane and probed with a rabbit antibody against human Helios overnight at 4°C. Proteins were visualized by chemiluminescence detection using HRP donkey anti-rabbit IgG (minimal x-reactivity) antibody (Cat. No. 406401) at a 1:3000 dilution. Lane M: Molecular weight marker.
Chromatin Immunoprecipitations (ChIP) were done with fixed and sonicated chromatin samples from Jurkat cells. ChIP was performed with 3 µg of chromatin and either 1.2 µg of Go-ChIP-Grade™ Purified Armenian Hamster IgG Isotype Control Antibody (clone HTK888, in black) or no antibody (beads control, in gray). Protein G beads were used to isolate immune complexes. The enriched DNA was purified and quantified by real-time qPCR using SYBR Green and primers for active (human GAPDH promoter and human RPL30 exon 3) and inactive (human α-Satellite repeats) loci. The amount of immunoprecipitated DNA in each sample is represented as percentage to the total amount of input chromatin, which is equivalent to 100%.
Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis as negative control, and the purity is greater than 95% by SDS-PAGE. Use at concentrations comparable to those of the specific antibody of interest. For immunoprecipitation, the suggested use of this reagent is 2.5 µg/test.
For in vivo studies or highly sensitive assays, we recommend Ultra-LEAF™ purified antibody (Cat. No. 400940) with a lower endotoxin limit (Endotoxin < 0.01 EU/µg).
Application References
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