Immunohistochemistry was performed on cancer biopsies of deparaffinized Human breast carcinoma tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing Calpastatin (MA3-944) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.
|Tested species reactivity||Bovine, Human, Pig, Rat|
|Published species reactivity||Bovine, Fish, Human|
|Host / Isotype||Mouse / IgG1|
|Immunogen||Purified bovine skeletal muscle 80 kDa subunit of m-Calpastatin.|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Western Blot (WB)||1:5,000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
|Western Blot (WB)||See 4 publications below|
MA3-944 detects calpastatin from bovine, human, pig and rat tissues and cells. This antibody does not cross-react with calpains or calmodulin.
MA3-944 has been successfully used in Western blot and immunocytochemical procedures. By Western blot, this antibody detects 150, 125, 90 and 70 kDa proteins from bovine skeletal muscle extract which have been suggested to be proteins translated from different start sites of the calpastatin gene. Immunocytochemical staining of calpastatin in porcine LLC-PK1 cells with MA3-944 results in diffuse cytoplasmic staining.
The MA3-944 antigen is purified bovine skeletal muscle 80 kDa subunit of m-calpastatin. This antibody recognizes an epitope between amino acid residues 543-673 (domain IV) of human calpastatin.
The calpain system has been detected in every vertebrate tissue examined, and has been suggested to play a regulatory role in cellular protein metabolism. This regulatory role may have important implications in platelet aggregation and pathologies associated with altered calcium homeostasis and protein metabolism such as ischemic cell injury and degenerative diseases. Inhibitors of calpain have been shown to block dexamethasone and low-level irradiation induced apoptosis in thymocytes suggesting that calpain has a regulatory or mechanistic role in apoptotic cell death.
Calpastatin, a specific endogenous inhibitor of calpain, has a predicted molecular weight of ~77 kDa and an internal repeat of four homologous domains which allow it to inhibit multiple calpain molecules simultaneously. It functions by binding to calpain when calpain binds calcium. When calpastatin is subjected to proteolytic digestion, fragments of calpastatin as small as 15 kDa still retain inhibitory activity.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Isolation and characterization of mu-calpain, m-calpain, and calpastatin from postmortem muscle. I. Initial steps.
MA3-944 was used in western blot to identify and characterize calpains from postmortem muscle
|Camou JP,Mares SW,Marchello JA,Vazquez R,Taylor M,Thompson VF,Goll DE||Journal of animal science (85:3400)||2007|
Purification and characterization of calpain and calpastatin from rainbow trout, Oncorhynchus mykiss.
MA3-944 was used in western blot to purify and characterize the calpain and calpastatin from rainbow trout
|Saito M,Li H,Thompson VF,Kunisaki N,Goll DE||Comparative biochemistry and physiology. Part B, Biochemistry and molecular biology (146:445)||2007|
The calpain system in human placenta.
MA3-944 was used in western blot to study the calpains present in human placenta
|Thompson VF,Saldaña S,Cong J,Luedke DM,Goll DE||Life sciences (70:2493)||2002|
Changes in the calpains and calpastatin during postmortem storage of bovine muscle.
MA3-944 was used in western blot to detect the changes of micro-calpain, m-calpain, and calpastatin in bovine muscle during postmortem storage
|Boehm ML,Kendall TL,Thompson VF,Goll DE||Journal of animal science (76:2415)||1998|