HeLa Cell Lysate, Heat Shocked

HeLa Cell Lysate (Heat Shocked) is useful in immunoblotting applications as a positive control for many antibodies, particularly those recognizing proteins induced by heat shock. The cells are grown, heat shocked for 2 hours at 44°C, and allowed to recover at 37°C for 5 to18 hours before harvest. The cells are then washed, lysed, sonicated, and resuspended in SDS-PAGE sample buffer. The HeLa cell line was originally derived from epithelioid carcinoma tissue (cervix) from a human donor. Research into the heat shock response provides a model for the study of the effects of environmental and homeostatic stressors upon cells. Heat shocking reproducibly alters gene expression in cells resulting in the subsequent production of a variety of morphological changes as well as the expression of heat shock proteins which are crucial for the maintenance of cellular homeostasis during normal cell growth and for survival during and after various cellular stresses. Heat shocking has been shown to increase morphological changes compatible with apoptosis in many cell types. In mammalian cells, the induction of the heat shock response requires the activation and nuclear translocation of transcription factors which control the expression of the cytoprotective heat shock proteins as well as playing pivotal roles in the development of inflammation and immunity. Applications: Suitable for use in Western Blot. Other applications not tested. Recommended Dilution: Western Blot: 20ug/lane, Optimal dilutions to be determined by the researcher. Production Method: HeLa cells were maintained in 90% Eagle's Minimal Essential medium with Earle's BSS, 0.1mM non-essential amino acids, 2mM L-glutamine, 10% fetal bovine serum, 50ug/ml gentamycin sulfate, 1mM sodium pyruvate. HeLa cells were heat shocked at 44°C for 2 hours and allowed to recover for five to eighteen hours at 37°C before harvesting. Cells were collected from media by centrifuging at 1200 rpm at 4°C in a Beckman GS-6R centrifuge for 7 minutes. Cells were washed twice in PBS and were lysed in M-PER Mammalian Protein Extraction Reagent (Pierce catalog# 78501) with a protease inhibitor cocktail. The cell lysate was adjusted to 2mg/mL in SDS PAGE sample buffer (40mM Tris-HCl pH 6.8, 1% SDS, 50mM DTT, 7.5% glycerol, 0.003% bromphenol blue) and heated or 10 min at 70ºC. Storage and Stability: Aliquot to avoid repeated freezing and thawing and freeze at -70°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Aliquots are stable for at least 6 months.