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Our Abpromise guarantee covers the use of ab2251 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 35 kDa (predicted molecular weight: 31 kDa).|
|IHC-Fr||Use at an assay dependent concentration. PubMed: 24223903|
|ICC/IF||Use a concentration of 10 µg/ml.|
|IHC-P||Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|IP||Use at an assay dependent concentration.|
IHC image of Caspase-2 staining in human hippocampus FFPE section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab2251, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
ICC/IF image of ab2251 stained A431 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2251, 10µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, anti-rabbit DyLight® 488 used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab2251 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.
Caspase 2 was immunoprecipitated using 0.5mg Jurkat whole cell extract, 5µg of Rabbit polyclonal to Caspase 2 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Jurkat whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab2251.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody anti-Rabbit IgG light chain HRP (ab99697).
Band: 35kDa: Caspase 2 .
ab2251 detects a band at 35 kDa in cell lysates and a 20 kDa band in the Caspase recombinant protein corresponding to the tagged form.