Immunohistochemistry was performed on cancer biopsies of deparaffinized Human breast carcinoma tissue. 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 1:1000 with a mouse monoclonal antibody recognizing Calpastatin (MA3-945) 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, Dog, Human, Pig, Rabbit, Rat|
|Published species reactivity||Dog, Rabbit, Bovine, Fish, Human|
|Host / Isotype||Mouse / IgG2a|
|Immunogen||Bovine skeletal muscle calpastatin.|
|Storage buffer||ascites diluted in PBS|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Immunohistochemistry (Frozen) (IHC (F))||1:200-1000|
|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.
MA3-945 detects calpastatin from bovine, human, canine, pig, rabbit, and rat tissues and cells. This antibody does not cross-react with calpains or calmodulin.
MA3-945 has been successfully used in Western blot, immunohistochemistry, and immunocytochemical procedures. By Western blot, this antibody detects 150, 125, 90 and 70 kDa proteins which have been suggested to be proteins translated from different start sites of the calpastatin gene. Immunofluorescence staining of calpastatin in porcine LLC-PK1 cells with MA3-945 results in diffuse cytoplasmic staining and weak nuclear staining.
The MA3-945 antigen is bovine skeletal muscle calpastatin. This antibody recognizes an epitope found between amino acid residues 295-501 (domain II) 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.
Postmortem proteolysis in three muscles from growing and mature beef cattle.
MA3-945 was used in western blot to study proteolysis and calpain/calpastatin in three postmortem muscles from young and mature beef cattle and the implications for meat tenderness
|Cruzen SM,Paulino PV,Lonergan SM,Huff-Lonergan E||Meat science (96:854)||2014|
Isolation and characterization of mu-calpain, m-calpain, and calpastatin from postmortem muscle. I. Initial steps.
MA3-945 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.
MA3-945 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|
Effect of nutrient restriction on calpain and calpastatin content of skeletal muscle from cows and fetuses.
MA3-945 was used in western blot to investigate the effect of nutrient restriction on calpain and calpastatin content of skeletal muscle from cows and fetuses
|Du M,Zhu MJ,Means WJ,Hess BW,Ford SP||Journal of animal science (82:2541)||2004|
Effect of dietary protein on calpastatin in canine skeletal muscle.
MA3-945 was used in western blot to investigate the role of dietary protein on the expression of calpastatin in canine skeletal muscle
|Helman EE,Huff-Lonergan E,Davenport GM,Lonergan SM||Journal of animal science (81:2199)||2003|
Subcellular localization and in vivo subunit interactions of ubiquitous mu-calpain.
MA3-945 was used in immunocytochemistry to study the subcellular localization and in vivo subunit interactions of mu-calpain
|Gil-Parrado S,Popp O,Knoch TA,Zahler S,Bestvater F,Felgenträger M,Holloschi A,Fernández-Montalván A,Auerswald EA,Fritz H,Fuentes-Prior P,Machleidt W,Spiess E||The Journal of biological chemistry (278:16336)||2003|
Fiber type-specific expression of major proteolytic systems in fast- to slow-transforming rabbit muscle.
MA3-945 was used in immunohistochemistry to study the importance of two major proteolytic systems in tranforming rabbit and rat muscles
|Sultan KR,Dittrich BT,Leisner E,Paul N,Pette D||American journal of physiology. Cell physiology (280:C239)||2001|