The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
ICC/IF: Use at a concentration of 1 µg/ml.
IHC-P: Use at a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
WB: 1/1000 when using colorimetric substrates - 1/5000 for chemiluminescent substrates. Predicted molecular weight: 30 kDa. Dilution optimised using Chromogenic detection. Not yet tested in other applications. Optimal dilutions/concentrations should be determined by the end user.
Regulatory subunit of the calcium-regulated non-lysosomal thiol-protease which catalyzes limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction.
Contains 5 EF-hand domains.
The contact of the 5th EF-hand domain from each monomer allows the formation of the homodimer and also appears to mediate the contact between the large catalytic subunit and small regulatory subunit for the formation of the heterodimer. EF-hand domains are paired. EF-hand 1 is paired with EF-hand 2 and EF-hand 3 is paired with EF-hand 4. The fifth EF-hand domain, left unpaired, does not bind the calcium but is responsible of the dimerization by EF-embrace. The first four EF-hand domains bind calcium, however it is not sure if the binding of EF-hand 4 to calcium is physiologically relevant.
Cytoplasm. Cell membrane. Translocates to the plasma membrane upon calcium binding.
ICC/IF image of ab28237 stained HeLa 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 (ab28237, 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.
Ab28237 staining Human normal renal medulla. Staining is localized the nucleus and cytoplasm. Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control. Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS), then incubated with primary antibody for 20 minutes, and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
Western blot - Anti-Calpain S1 antibody (ab28237)Image from Su SC et al, J Biol Chem. 2010 Dec 24;285(52):40624-34. Epub 2010 Oct 13, Fig 2.
Plasma membrane (Membrane) and cytoplasmic (Cytosol) fractions were prepared from HUVEC cells and immunoblotted with antibodies against the indicated proteins. HRP-conjugated secondary antibodies were used. Blots are representative of four independent experiments.
Lin YW et al. Receptor-Interacting Protein 140 Orchestrates the Dynamics of Macrophage M1/M2 Polarization. J Innate Immun8:97-107 (2016).
Read more (PubMed: 26228026) »
Ozaki T et al. Intravitreal injection or topical eye-drop application of a µ-calpain C2L domain peptide protects against photoreceptor cell death in Royal College of Surgeons' rats, a model of retinitis pigmentosa. Biochim Biophys Acta1822:1783-95 (2012).
Read more (PubMed: 22885154) »