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#2305-PC-020 Anti_Cleaved_Caspase_3 PC Sample
Product name : Anti_Cleaved_Caspase_3 PC Sample
Catalog number : 2305-PC-020
Quantity: 20 µl
Supplier name : Trevigen
Data sheet: Ask more or other datasheet now !
More Details about
Anti-Cleaved Caspase-3 Rabbit Polyclonal Antibody
Catalog #: 2305-PC-020
Size: 20 μl
Description: Rabbits were immunized with the KLH-coupled synthetic peptide CRGTELDCGIETD. The polyclonal antibody was affinity purified on a column derivatized with the peptide. It detects the processed large subunit (p18 fragment) but not the unprocessed, zymogen form of caspase-3. In apoptotic lysates, the antibody may recognize the induction of a strong p30 band (Fig. 1).
Physical State: This antibody is provided as affinity purified IgG fraction in 1X PBS without preservative. The final antibody concentration is 0.1 mg/ml.
Immunogen: 13-amino acid peptide sequence from carboxyl terminus of the human and mouse caspase-3 p18 subunit.
Specificity: The antibody detects human and mouse cleaved caspase-3.
Storage: The antibody can be stored at 4°C for at least 1 month, or can be aliquoted and stored frozen at -20°C to -80°C. Avoid repeated freezing and thawing by aliquoting into smaller portions.
Applications: For immunodetection, an antibody dilution of 1:500-1:1000 is recommended. May also be used for immunoprecipitation and immunocytochemistry at a 1:100-1:1000 dilution. Empirical determination will be required for optimal results.
Reference: 1. Srinivasan, A., K.A. Roth, R.O. Sayers, K.S. Shindler, A.M. Wong, L.C. Fritz, and K.J. Tomaselli. 1998. In situ immunodetection of activated caspase-3 in apoptotic neurons in the developing nervous system. Cell Death Diff 5:1004-1016.
Fig 1. Immunoblot of SDS-extracts from mouse cells treated with 25 μM etoposide. Samples were electrophoresed on a 12% Tris-Glycine gel and blotted onto a PVDF membrane. The 18 kDa cleaved caspase-3 was detected by Trevigen’s anti-cleaved caspase-3 followed by anti-rabbit conjugated to horseradish peroxidase and chemiluminescence.
Fig. 2. Jurkat cells were treated with 25 μM etoposide for 5 hours to induce apoptosis. Cells were labeled with 1:250 dilution of anti-cleaved caspase-3 followed by anti-rabbit horseradish peroxidase. Detection was performed with Trevigen’s Blue Label and counterstained with Nuclear Fast Red.
Cell Lysates for Western Blotting:
To prepare total cell lysates, cells are solubilized in 1X SDS gel sample buffer (20 mM dithiothiothreitol, 6% SDS, 250 mM Tris, pH 6.8, 10% glycerol, and bromophenol blue) at 5 x 105 - 1 x 106 cells per ml. The extracts are heated in a boiling water bath for 5 minutes. Electrophorese on 12% Tris-Glycine SDS-PAGE gels.
Procedure for Immunoblotting using Peroxidase Detection:
Blotting buffer: 12 mM Tris base, 96 mM Glycine, and 20% MeOH.
Blocking solution: 5% (w/v) nonfat dry milk in PBS.
Antibody solution: 5% (w/v) nonfat dry milk, 0.05% Tween in PBS.
Transfer the electrophoresed proteins to a PVDF membrane and incubate the membrane for 1/2 hour at room temperature in blocking solution. Incubate the membrane overnight at 4°C in antibody solution containing a 1:500 -1:1000 dilution of cleaved caspase-3 rabbit polyclonal antibody. Empirical determination of primary antibody concentration will be required for optimal results. Wash the membrane at room temperature for 15 minutes with 3 changes of 0.05% Tween in PBS. Changing membrane containers often reduces background. Incubate the membrane at room temperature for 1 hour in an antibody solution containing anti-rabbit IgG conjugated to horseradish peroxidase. Empirical determination of secondary antibody concentration will be required for optimal results. Wash the membrane for 15 minutes with 3 changes of 0.05% Tween in PBS. Develop peroxidase reaction using e.g. chemiluminescence.
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