WI-38 nuclear extract (Forskolin stimulated)

Contents

2 x 100 µg of WI-38 nuclear extract (Forskolin stimulated) at 2.5 µg/µl.

Background

WI-38 nuclear extract (Forskolin stimulated) was prepared from a cell culture of the human lung fibroblast WI-38 cell line. The WI-38 cell line was originally derived from lung tissue obtained from a Caucasian female fetus of 3 months gestation. The WI-38 cell line was the first normal diploid human cell line to be continuously cultivated. WI-38 cells are known to have the broadest viral spectra of any cell population that has been tested and are frequently used in research to isolate viruses, such as rhinovirus, and for the production of viral vaccines, including rubella, rabies, and hepatitis A. These cells have a limited replicative lifespan of approximately 50 population doublings after which they enter a state of irreversible growth arrest known as replicative senescence. WI-38 cells are most commonly used in research in studies of viral production, replicative senescence, and as normal controls.

Forskolin is an inducer of intracellular cyclic AMP (cAMP), a second messenger that is used for signal transduction during various biological processes, including lipid metabolism and regulation of glycogen and sugar levels. Forskolin was used to stimulate cAMP production in WI-38 cells, leading to the downstream activation of protein kinase A (PKA) and calcium channels.

Application Notes

WI-38 nuclear extract (Forskolin stimulated) is specifically recommended for studies related to cAMP-mediated signal transduction.

Extract Origin

Human normal lung fibroblast

Extract Composition

WI-38 nuclear extract was collected in Lysis Buffer after a 30-minute incubation with forskolin (20 µM). The Lysis Buffer consists of 20 mM Hepes pH 7.5, 400 mM NaCl, 20% glycerol, 0.1 mM EDTA, 10 mM NaF, 10 µM Na₂MoO₄, 1 mM NaVO₃, 10 mM PNPP, 10 mM β-glycerophosphate, 1 mM DTT and protease inhibitors. The protein content has been determined by a Bradford-based assay.

Quality Control

Each lot has been tested for CREB activation by using TransAM^® pCREB Kits. The signal intensity for CREB activation in each lot is compared to the signal intensity obtained with extracts from unstimulated WI-38 VA13 cells (see figure). After the signals are blanked, the ratio of the signals from stimulated cells over unstimulated cells must be above 3. This ratio may vary depending on the basal level of CREB activation in a given cell type.

Storage

To ensure stability, extracts should be stored at -80°C.

We recommend aliquoting the extracts into single-use fractions and then storing them at -80°C. This eliminates repeated freeze/thaw cycles.

Guarantee

This product is guaranteed for 6 months from date of receipt.

This product is for research use only and is not for use in diagnostic procedures.