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.
WB: 1/1000 when using colorimetric detection system (BCIP/NBT substrates); 1/5000 when using chemoluminescent substrates. Predicted molecular weight: 76 kDa. But note that when used against the reduced protein identifies bands at 70 Kd, 52 Kd, and a series of further cleaved active forms. Dilution optimised using Chromogenic detection. Not yet tested in other applications. Optimal dilutions/concentrations should be determined by the end user.
Specific inhibition of calpain (calcium-dependent cysteine protease). Plays a key role in postmortem tenderization of meat and have been proposed to be involved in muscle protein degradation in living tissue.
Belongs to the protease inhibitor I27 (calpastatin) family.
Immunocytochemistry/ Immunofluorescence-Calpastatin antibody - Aminoterminal end (Domain L) Testis Isoform(ab28252)
ICC/IF image of ab28252 stained HeLa 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 (ab28252, 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 - Calpastatin antibody - Aminoterminal end (Domain L) Testis Isoform (ab28252)This image is courtesy of an anonymous Abreview.
All lanes : Anti-Calpastatin antibody (ab28252) at 1/5000 dilution
All lanes : Mouse skeletal tissue muscle lysate
Lysates/proteins at 30 µg per lane.
Secondary All lanes : HRP-conjugated Goat IgG to Rabbit
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Kemp CM et al. The effects of Capn1 gene inactivation on skeletal muscle growth, development, and atrophy, and the compensatory role of other proteolytic systems. J Anim Sci91:3155-67 (2013).
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