ab28257 recognizes the amino terminal end 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, it does not cross react with the other calpain family members (M Calpain, Calpain 94, ncl2, ncl3, etc.).
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/1000 - 1/5000. Detects a band of approximately 82 kDa (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, ab28257 identifies bands at 80 kDa, 68 kDa, and a series of further cleaved forms. Dilution optimised using Chromogenic detection.
Use a concentration of 1 - 5 µg/ml.
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.
ICC/IF image of ab28257 stained MCF7 cells. The cells were 100% methanol fixed (5 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 (ab28257, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 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.
Western blot - Anti-Calpain 1 antibody (ab28257)
All lanes : Anti-Calpain 1 antibody (ab28257) at 1 µg/ml
Lane 1 : Human liver tissue lysate - total protein (ab29889) Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 3 : U2OS (Human osteosarcoma cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 82 kDa Observed band size: 82 kDa Additional bands at: 27 kDa, 42 kDa, 65 kDa. We are unsure as to the identity of these extra bands.
Scott IC et al. Interleukin-33 is activated by allergen- and necrosis-associated proteolytic activities to regulate its alarmin activity during epithelial damage. Sci Rep8:3363 (2018).
Read more (PubMed: 29463838) »
Tan WJ et al. Calpain 1 regulates TGF-ß1-induced epithelial-mesenchymal transition in human lung epithelial cells via PI3K/Akt signaling pathway. Am J Transl Res9:1402-1409 (2017).
Read more (PubMed: 28386365) »