ab28258 recognizes Domain I of the large subunit of Calpain 1. The antibody does not recognize amino-processed Calpain 1, and can be used to discriminate between latent and amino-processed forms. Also ab28258 does not recognize the other calpain family members (M calpain, calpain 94, ncl 2, ncl 3, etc.).
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/1000 - 1/5000. Predicted molecular weight: 82 kDa. 1/1000, when using colorimetric substrates such as BCIP/NBT - 1/5000, when using chemiluminescent substrates. But note that when used against the reduced protein, ab28258 identifies bands at 80 kDa, 68 kDa, and a series of further cleaved forms. Dilution optimised using Chromogenic detection.
Use at an assay dependent concentration.
ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.
Calcium-regulated non-lysosomal thiol-protease which catalyze limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction.
Belongs to the peptidase C2 family. Contains 1 calpain catalytic domain. Contains 4 EF-hand domains.
Cytoplasm. Cell membrane. Translocates to the plasma membrane upon Ca(2+) binding.
Immunocytochemistry/ Immunofluorescence - Anti-Calpain 1 antibody (ab28258)This image is courtesy of an anonymous Abreview
ab28258 staining human HUVEC cells by ICC/IF. Cells were PFA fixed and permeablized in 0.5% Triton X-100 prior to blocking in 1% serum for 16 hours at 4°C. The primary antibody was diluted 1/100 and incubated with the sample for 2 hours at 25°C. A FITC conjugated goat anti-rabbit antibody, diluted 1/100, was used as the secondary.
Flow Cytometry - Anti-Calpain 1 antibody (ab28258)This image is courtesy of an abreview submitted by Mahesh Shivananjappa.
ab28258 detecting Calpain 1 - Domain I in Human platelets by Flow Cytometry. Platelets were isolated by spinning Platelet rich plasma on Histopaque. Cells were fixed in PFA and permeabilized in 0.1% Triton-X100 in 2% BSA for 30 minutes. The primary antibody was diluted 1/250 in 2% BSA in PBS and incubated with the sample for 18 hours at 4°C. An Alexa Fluor® 488-conjugated Chicken anti-Rabbit IgG (H+L) antibody, diluted 1/500 was used as the secondary antibody.
Abbreviations in the figure:
US Unstained, Red Peak;
IgG Rb: IgG Rabbit (Isotype Control), Blue Peak;
Calpain: Green Peak.
Pahima H et al. Hypoxic-induced truncation of voltage-dependent anion channel 1 is mediated by both asparagine endopeptidase and calpain 1 activities. Oncotarget9:12825-12841 (2018).
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