Western blot analysis of NIH/3T3 and HeLa cells, untreated (-) or treated with Staurosporine #9953 (1 μM, 3 hr; +), using Cleaved Caspase Substrate Motif [DE(T/S/A)D] MultiMab™ Rabbit mAb mix (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 as a loading control (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 263
Cleaved Caspase Substrate Motif [DE(T/S/A)D] MultiMab™ Rabbit mAb mix recognizes endogenous levels of caspase-cleaved proteins with a carboxy-terminal aspartic acid residue, and in rare cases a carboxy-terminal glutamic acid residue . This antibody does not cross-react with whole proteins or those ending with a different carboxy-terminal residue.Species Reactivity:
All Species Expected
MultiMab™ rabbit monoclonal mix antibodies are prepared by combining individual rabbit monoclonal clones in optimized ratios for the approved applications. Each antibody in the mix is carefully selected based on motif recognition and performance in multiple assays. Each mix is engineered to yield the broadest possible coverage of the modification being studied while ensuring a high degree of specificity for the modification or motif.
Apoptosis is a physiological process resulting in a highly regulated, programmed form of cell death that is a normal part of growth and development in multicellular organisms. Caspases are aspartic acid-directed cysteine proteases that are central to the apoptotic mechanism (1). The intrinsic pathway initiates an apoptotic cascade from signals originating within the cell, such as DNA damage, while an extrinsic pathway initiates apoptosis in response to extracellular signals, like FasL. In both intrinsic and extrinsic pathways, initiator caspases cleave downstream substrates, including multiple effector caspases and the primary executioner of cell death, caspase-3 (2,3). Effector caspases amplify the apoptotic cascade to target many critical proteins needed for normal cell function. Apart from its role in developmental biology, the regulation of apoptosis has broad implications for the study of cancer, autoimmune disorders and infectious diseases (4). Thousands of known and putative caspase cleavage sites are present within the human proteome; almost all sites involve cleavage at an aspartic acid residue, though cleavage at glutamic acid residues is seen, rarely, as well (5).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
MultiMab is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
Use of Cell Signaling Technology (CST) Motif Antibodies within certain methods (e.g., U.S. Patents No. 7,198,896 and 7,300,753) may require a license from CST. For information regarding academic licensing terms please have your technology transfer office contact CST Legal Department at CST_ip@cellsignal.com. For information regarding commercial licensing terms please contact CST Pharma Services Department at firstname.lastname@example.org.