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Methods for detecting membrane derived caspase activity and modulators thereof

A technology of activity and source, applied in the direction of biochemical equipment and methods, measuring devices, microbial determination/testing, etc., can solve the problems of lack of specificity, effectiveness and practicability, method limitations, etc.

Inactive Publication Date: 2002-04-17
IDUN PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Available methods are limited due to lack of specificity, efficacy, and / or utility (Utilization) of intact cells or their cytoplasmic extracts

Method used

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  • Methods for detecting membrane derived caspase activity and modulators thereof
  • Methods for detecting membrane derived caspase activity and modulators thereof
  • Methods for detecting membrane derived caspase activity and modulators thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Cell Lines and Cell Culture

[0096]Stable transfections containing Bcl-2 cDNA (697-Bcl-2 cells) or control neomycin resistance gene (697-neo cells) (Miyashita and Reed, 1993) (obtained from Dr. John Reed , Burnham Institute) retroviral expression constructs of 697 human lymphoblastoid cells. These cells were treated with 10% fetal bovine serum ((FBS)Hyclone, Logan, UT), 0.2 mg / ml G-418 (Gibco, Gaithersburg, MD) and 0.1 mg / ml penicillin / streptomycin (Irvine Scientific, Growth in mid-log phase was maintained in RPMI 1640 medium (Irvine Scientific) in Santa Aha, Ca). Murine dopaminergic MN9D cells (obtained from Dr. A. Heller, University of Chicago) were cultured in DMEM medium (Irvine Scientific) supplemented with 10% FBS, 2 mM glutamine and 0.1 mg / ml penicillin / streptomycin. Pregnant E15 mouse cerebral cortex cells were prepared in Hank's buffered saline solution (Irvine Scientific) containing 15 mM HEPES. The tissue was briefly dissociated with 0.1% trypsin, then th...

Embodiment 2

[0098] subcellular fractionation

[0099] will contain ≈10 9 Frozen cell pellets of two cells were thawed and resuspended in supplemented with 1 mM PMSF, 1 μg / ml leupeptin, 1 μg / ml pepstatin A, 5 μg / ml aprotinin, 0.1 mM EDTA, 0.1 mM EGTA and 5mM DTT (Sigma) in cold hypotonic buffer (10mM Na-HEPES, 5mMMgCl 2 , 42mM KCl, pH 7.4), the density is ≈1.5×10 8 cells / ml. The samples were incubated on ice for 30 minutes during which time the cells were lysed with 30-40 strokes of a Dounce homogenizer. The samples were centrifuged twice at 500 xg for 10 minutes at 4°C to isolate cell nuclei. The nuclear pellet was then washed twice in the same buffer supplemented with 1.6M sucrose to yield the nuclear fraction. The supernatant from the above centrifugation step was then centrifuged at 14,000×g, 4°C for 30 minutes to pellet the heavy membrane. The heavy membrane was washed 3 times with 1.5 ml of cold hypotonic buffer containing protease inhibitors and DTT. Washed membranes were res...

Embodiment 3

[0101] western blot

[0102] Subcellular fractions (50 μg protein per lane) were separated by SDS-PAGE on 12% or 16% gels (Novex, La Jolla, CA) and then transferred to Immobilon PVDF membranes (Millipore, Bedford, MA) . Membranes were blocked in PBS / 0.1% Tween 20 (PBST) + 0.4% casein (I-block, Tropix, Bedford, MA). Incubate blots for 1 hr in 1 μg / ml primary antibody diluted in PBST / casein. After 3 washes in PBST, blots were incubated for 1 hour in alkaline phosphatase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Tropix) diluted 1:15,000 in PBST / casein. The blot was then washed twice with PBST in assay buffer (10 mM diethanolamine, pH 10.0, 1 mM MgCl 2 ), then incubated with 250 μM chemiluminescence substrate CSPD (Tropix) in assay buffer and exposed to Biomax film (Kodak, Rochester, NY) overnight.

[0103] In some cases, after incubation with the secondary antibody, 10 mM Tris, pH 9.5, 1 mM MgCl 2 Wash blot. The blot was then incubated for 30 minutes in 1.25 μ...

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Abstract

The present invention provides methods for detecting membrane-derived apoptotic activity. In one embodiment, the present invention provides methods of identifying membrane-derived caspase activity. In another embodiment, drug discovery methods are provided for screening compounds that inhibit or enhance the activity of membrane-derived caspases. In these various embodiments, heavy membrane fractions are used in the screening methods described herein.

Description

field of invention [0001] In general, the present invention relates to methods for detecting membrane-derived caspase activity and modulators thereof, and more particularly, to a novel cell-free screening assay for identifying inhibitors and enhancers of membrane-derived caspase activity. Background of the invention [0002] Tissue homeostasis is maintained through the process of apoptosis, the normal physiological process of programmed cell death. As with abnormalities in cell cycle regulation, alterations in apoptotic pathways that prevent or delay normal cell turnover are often important in the pathogenesis of disease. Just as cell division is controlled through complex interactions between cell cycle regulatory proteins, apoptosis is normally regulated through the interaction of gene products that act to prevent or induce cell death. [0003] Since apoptosis plays a role in maintaining tissue homeostasis in a variety of physiological processes, such as embryonic develop...

Claims

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Application Information

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IPC IPC(8): C12Q1/37
CPCC12Q1/37G01N2333/96466C12Q1/00
Inventor L·C·福里茨J·F·克雷博斯
Owner IDUN PHARMA INC
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