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> Chemicals & Drugs > Amino Acid > Calpain

Calpain

Calpains are a family of calcium-activated, non-lysosomal cysteine proteases involved in a variety of cellular processes.
They play a role in cell signaling, cytoskeletal remodeling, and apoptosis.
Calpains have been implicated in the pathogenesis of numerous diseases, including neurodegeneration, muscular dystrophy, and cancer.
Reasearching optimal experimental protocols for studying calpain activity and regulation is crucial for advancing our understanding of this important protease family and developing potential therapeutics.
PubCompare.ai's AI-powered technology can help researchers quickly identify the most reproducible and effective calpain research protocols from the literature, preprints, and patents to accelerate their investigations.

Most cited protocols related to «Calpain»

1
Immunoblot Analysis of α-Synuclein and Synaptic Markers
Cited 12 times
Briefly, as previously described, brains were homogenized and divided into cytosolic and membrane fractions [12] , [15] (link). For immunoblot analysis, 20 µg of total protein per lane was loaded into 4–12% Bis-Tris SDS-PAGE gels and blotted onto polyvinylidene fluoride (PVDF) membranes. For characterization of the antibodies samples from untreated non-tg and α-syn tg mice were incubated the with monoclonal antibodies against CT and N-Terminal (NT) α-syn (9E4, 6H7 and 8A5, ELAN Pharmaceuticals). To determine the effects of the immunotherapy in levels of α-syn blotted samples from treated α-syn tg were probed with antibodies against calpain-cleaved α-syn (CC α-syn) which recognizes a C-terminal fragment of α-syn [32] (link), full length α-syn (FL α-syn rabbit polyclonal (1∶1000, Millipore, Temecula CA). For the analysis of synaptic proteins, monoclonal antibodies against Synapsin I (1∶1000, Millipore, Temecula, CA) and PSD95 (UC Davis/NIH Neuro-Monoclonal Antibody Facility, Davis, CA) were used. In order to determine the effects of the immunotherapy on levels of total tau and PHF-tau blotted samples from treated α-syn tg were probed with antibodies against total tau (1∶1000, Dako, Carpinteria, CA) and PHF-tau (1∶1000, UC Davis/NIH Neuro-Monoclonal Antibody Facility, Davis, CA). Incubation with primary antibodies was followed by species-appropriate incubation with secondary antibodies tagged with horseradish peroxidase (1∶5000, Santa Cruz Biotechnology, Santa Cruz, CA), visualization with enhanced chemiluminescence and analysis with a Versadoc XL imaging apparatus (BioRad, Hercules, CA). Analysis of β-actin (Sigma) levels was used as a loading control.
Masliah E., Rockenstein E., Mante M., Crews L., Spencer B., Adame A., Patrick C., Trejo M., Ubhi K., Rohn T.T., Mueller-Steiner S., Seubert P., Barbour R., McConlogue L., Buttini M., Games D, & Schenk D. (2011). Passive Immunization Reduces Behavioral and Neuropathological Deficits in an Alpha-Synuclein Transgenic Model of Lewy Body Disease. PLoS ONE, 6(4), e19338.
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Publication 2011
Actins Antibodies Bistris Brain Calpain Chemiluminescence Cytosol Gels Horseradish Peroxidase Immunoblotting Immunotherapy MAPT protein, human Mice, House Monoclonal Antibodies Pharmaceutical Preparations polyvinylidene fluoride Proteins Rabbits SDS-PAGE Synapsin I Tissue, Membrane
2
Calpain Substrate Cleavage Site Identification
Cited 9 times
We searched the scientific literature from PubMed with the keyword of “calpain” to obtain the experimentally verified calpain substrates with cleavage sites (before June 30th, 2010). The data collected by Tompa et al. and duVerle et al. were also integrated [16] (link), [22] (link), while the protein sequences were retrieved from the UniProt database.
We defined a calpain cleavage peptide CCP(m, n) as a cleavage bond flanked by m residues upstream and n residues downstream. As previously described [23] (link), [24] (link), we regarded all experimentally verified cleavage sites as positive data (+), while all other non-cleavage sites in the same substrates were taken as negative data (−). If a cleavage site locates at the N- or C-terminus of the protein and the length of the peptide is smaller than m+n, we added one or multiple “*” characters as pseudo amino acids to complement the CCP(m, n). The positive data (+) set for training might contain several homologous sites from homologous proteins. If the training data were highly redundant with too many homologous sites, the prediction accuracy would be overestimated. To avoid such overestimation, we clustered the protein sequences with a threshold of 40% identity by CD-HIT [25] (link). If two proteins were similar with ≥40% identity, we re-aligned the proteins with BL2SEQ, a program in the BLAST package [26] (link), and checked the results manually. If two calpain cleavage sites from two homologous proteins were at the same position after sequence alignment, only one item was preserved, the other was discarded. Finally, the non-redundant benchmark data set for training contained 368 positive sites from 130 unique substrates (Supplementary Table S1).
Liu Z., Cao J., Gao X., Ma Q., Ren J, & Xue Y. (2011). GPS-CCD: A Novel Computational Program for the Prediction of Calpain Cleavage Sites. PLoS ONE, 6(4), e19001.
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Publication 2011
Amino Acids Amino Acid Sequence Calpain Character Cytokinesis Peptides Protein C Proteins Proteolysis Sequence Alignment
3
Molecular Mechanisms of Cardioprotection
Cited 7 times
A detailed Methods section is available in the Online Supplement at http://circres.ahajournals.org, which includes the description of animal models, ex vivo global ischemia/reperfusion, in vivo regional ischemia, rat myocytes isolation and virus infection, mouse cardiomyocyte isolation and calcium kinetics measurement, cardiomyocyte apoptosis treatment and Annexin V staining, western blot analysis, caspase-3 and calpain activities measurement, DNA fragmentation measurement, terminal dUTP nick end labeling assays, plasma troponin I measurement, quantitative real-time PCR assay, generation of recombinant protein and blot overlay assay, GST-pull down assay, competitive protein binding ELISA assay, co-immunoprecipitation, immuno-fluorescence staining and statistical analysis.
Lam C.K., Zhao W., Cai W., Vafiadaki E., Florea S.M., Ren X., Liu Y., Robbins N., Zhang Z., Zhou X., Min J., Rubinstein J., Jones W.K, & Kranias E.G. (2012). Novel Role of HAX-1 in Ischemic Injury Protection: Involvement of Hsp90. Circulation research, 112(1), 79-89.
Publication 2012
Animal Model Annexin A5 Apoptosis Biological Assay Calcium Calpain Caspase 3 Co-Immunoprecipitation deoxyuridine triphosphate Dietary Supplements DNA Fragmentation Enzyme-Linked Immunosorbent Assay Immunofluorescence Ischemia isolation Kinetics Lanugo Mice, House Muscle Cells Myocytes, Cardiac Plasma Proteins Real-Time Polymerase Chain Reaction Recombinant Proteins Reperfusion Troponin I Virus Diseases Western Blot
4
Computational Modeling of Mini-Calpain-5 Structure
Cited 5 times

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Publication 2016
2-Mercaptoethanol Calcium Calpain CAPN9 protein, human Catalysis Catalytic Domain Hybrids leupeptin Mental Orientation Sequence Alignment
5
Neuromuscular Disease Patient Muscle Biopsy Profiling
Cited 5 times
Flash-frozen neuromuscular disease patient muscle biopsies were taken for diagnostic purposes. Normal control biopsies were obtained from baseline samples from exercise experiments in young adult volunteers.
Two datasets were analyzed. The first (test set) has been previously published (Bakay et al., 2006 (link)). This set contained Affymetrix mRNA profiles from 117 patient muscle biopsies using both HG-U133A and HG-U133B microarrays (n = 234 microarrays total; GEO accession no. GSE3307). The muscle disease groups were DMD (n = 10), amyotrophic lateral sclerosis (ALS; n = 9), acute quadriplegic myopathy (n = 5), BMD (n = 5), CAL (calpain III gene mutations; LGMD2A; n = 10), DYSF (LGMD2B; n = 10), ED-R (Emery–Dreifuss muscular dystrophy X linked; n = 4), ED-D (n = 4), FKRP (LGMD2I; n = 7), facioscapulohumeral dystrophy (FSH; n = 14), JDM (n = 21), and normal human skeletal muscle (NHM; n = 18). Biopsies were taken generally at the time of diagnosis and were typically from the vastus lateralis. The original published dataset analyzed 13 groups, but we removed the hereditary spastic paraplegia group from analyses reported here because of a high proportion of outlier transcripts and also removed four JDM samples that were not made public. To generate the mRNA profiles, frozen muscle biopsies were solubilized, total RNA was isolated, RNA was converted into biotinylated cRNA probes, hybridized to Affymetrix microarrays, and detected using fluorescent streptavidin, and microarrays were scanned. Quality control metrics used were as previously reported (Tumor Analysis Best Practices Working Group, 2004 (link)).
A second set of 49 human patient mRNA profiles was generated using HG-U133 Plus 2.0 microarrays, and this dataset is new to this study. These datasets contained profiles from 6 normal controls, 17 DMD (absence of dystrophin), 11 BMD (present but abnormal dystrophin), 7 LGMD2I (FKRP deficiency, a glycosylation defect), and 8 LGMD2B (DYSF). Patients had a broad range of ages, clinical severity of their disease, and histopathological findings, although all neuromuscular disease patients showed evidence of a dystrophic process (degeneration/regeneration of muscle fibers). Hematoxylin and eosin stains were performed on frozen sections, and the amount of fibrotic replacement (fibrosis) visually approximated for by the same evaluator (E.P. Hoffman) into normal, mild, moderate, or severe fibrosis categories.
Dadgar S., Wang Z., Johnston H., Kesari A., Nagaraju K., Chen Y.W., Hill D.A., Partridge T.A., Giri M., Freishtat R.J., Nazarian J., Xuan J., Wang Y, & Hoffman E.P. (2014). Asynchronous remodeling is a driver of failed regeneration in Duchenne muscular dystrophy. The Journal of Cell Biology, 207(1), 139-158.
Publication 2014
Amyotrophic Lateral Sclerosis Biopsy Calpain Complementary RNA Diagnosis DMD protein, human Eosin Fibrosis Freezing Frozen Sections Genes Hematoxylin Homo sapiens LGMD2I Limb-girdle muscular dystrophy, type 2B Limb-girdle muscular dystrophy type 2A Microarray Analysis Muscle Tissue Muscular Dystrophy, Emery-Dreifuss Muscular Dystrophy, Facioscapulohumeral Mutation Myopathy Neoplasms Neuromuscular Diseases Patients Protein Glycosylation Regeneration RNA, Messenger Skeletal Muscles Spastic Paraplegia, Hereditary Staining Streptavidin Vastus Lateralis Voluntary Workers Young Adult

Most recents protocols related to «Calpain»

1
Measuring Muscle Myofibril Fragmentation
As an indirect measure of calpain activity, myofibril fragmentation (MFI) in muscle samples was assessed. A total of 4 g of minced muscles, free of visible connective tissue and external fat, were homogenized for 30 s in a blender (Ultra Turrax; IKA-Werke, Staufen, Germany) with 40 ml of cold MFI buffer at 2°C. Following several washes, suspension aliquots were diluted in MFI buffer to a final protein concentration of 0.5 mg/ml and poured into a cuvette for immediate absorbance measurement at 540 nm with a spectrophotometer (HACH DR/3000 Spectrophotometer, USA). Each sample's MFI was multiplied by 200.
Suliman G.M., Hussein E.O., Alsagan A., Al-Owaimer A.N., Alhotan R., Al-Baadani H.H., Ba-Awadh H.A., Qaid M.M, & Swelum A.A. (2023). Effects of adding nano-emulsified plant oil and probiotics to drinking water during different periods besides sex on processing characteristics, physicochemical properties, and meat quality traits of broiler chickens. Frontiers in Veterinary Science, 10, 1133605.
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Publication 2023
Buffers Calpain Cold Temperature Connective Tissue Muscle Tissue Myofibrils Staphylococcal Protein A
2
Regulation of Sperm ROS Production
ROS production in guinea pig spermatozoa was assessed under non-capacitive and capacitive conditions, with or without specific NOX inhibitor, VAS2879 (40 µM), a pan-NOX inhibitor [27 (link)]; ML171 (2 µM) or NSC23766, a small GTPase Rac1 inhibitor (100 µM). We also tested the effect of calpain inhibition on ROS production using calpeptin (10 µM), a calpain-specific inhibitor.
Ortiz-García C.I., Salgado-Lucio M.L., Roa-Espitia A.L., Muñoz-Sánchez A.A., Cordero-Martínez J, & Hernández-González E.O. (2023). Calpain Regulates Reactive Oxygen Species Production during Capacitation through the Activation of NOX2 and NOX4. International Journal of Molecular Sciences, 24(4), 3980.
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Publication 2023
Calpain calpain inhibitor calpeptin Cavia ML171 Monomeric GTP-Binding Proteins NSC 23766 Psychological Inhibition Sperm
3
Fluorometric Calpain Activity Assay
The activated calpain in the cytosol of incubated erythrocytes was fluorometrically determined using a calpain activity assay kit according to the manufacturer’s instruction (Genway Biotech, San Diego, CA, USA).
Ferdous Z., Elzaki O., Beegam S., Zaaba N.E., Tariq S., Adeghate E, & Nemmar A. (2023). Comparative Evaluation of the Effects of Amorphous Silica Nanoparticles on the Erythrocytes of Wistar Normotensive and Spontaneously Hypertensive Rats. International Journal of Molecular Sciences, 24(4), 3784.
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Publication 2023
Biological Assay Calpain Cytosol Erythrocytes
4
Erythrocyte Exposure to Silica Nanoparticles
The erythrocytes were prepared using a previously described method [36 (link),68 (link)]. Briefly, collected rat blood was mixed by gentle inversion of the tube and centrifuged at 1200× g for 15 min. The plasma supernatant was discarded, and the erythrocytes were washed 4 times by suspending them in 0.9% NaCl followed by centrifugation at 1200× g for 10 min. The final suspension consisted of 5% by volume of erythrocyte in saline. Flat bottom 12 well plates were used to incubate 900 µL of erythrocyte with 100 µL of SiNPs (0.2, 1, 5, and 25 µg/mL). The plate was positioned in an MS 3 digital microtiter shaker (IKA WER KE GmbH & CO, Staufen, Germany) and rotated at 300 rpm for 30 min at room temperature, under shaded light. After incubation, the samples were transferred to a 1.5 mL Eppendorf tube and were centrifuged at 1200× g for 5 min. The resulting supernatant was collected for hemolysis assay, oxidative markers assays, intracellular calcium, Annexin V binding, ATPase activity, and calpain activity. The deposited erythrocytes were fixed with Karnovsky’s fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer at 7.2 pH) for transmission electron microscopy (TEM). The deformed erythrocytes were quantitated and expressed as the number of RBCs per field at a magnification of 11,100.
Ferdous Z., Elzaki O., Beegam S., Zaaba N.E., Tariq S., Adeghate E, & Nemmar A. (2023). Comparative Evaluation of the Effects of Amorphous Silica Nanoparticles on the Erythrocytes of Wistar Normotensive and Spontaneously Hypertensive Rats. International Journal of Molecular Sciences, 24(4), 3784.
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Publication 2023
Adenosinetriphosphatase Annexin A5 Biological Assay BLOOD Buffers Calcium Calpain Centrifugation Erythrocytes Fixatives Glutaral Hemolysis Inversion, Chromosome Light Normal Saline paraform Phosphates Plasma Protoplasm Saline Solution Transmission Electron Microscopy Volume, Erythrocyte
5
Fluorogenic Assays for Protease Inhibitors
The 3CLpro (SARS-CoV-2) assay kit (BPS Bioscience, San Diego CA, cat. no. 78042-2) is designed to measure 3CLpro activity and identify inhibitors of this enzyme, while human cathepsin L inhibitor screening kit (abcam, cat. Cambridge, UK, No. ab197012) and human calpain activity assay kit (AnaSpec. Inc. Fremont CA, cat. No. AS-72149) are designed to measure human cysteine protease activity and identify inhibitors of these enzymes. These assays were performed in a 96-well plate using a fluorogenic substrate. Briefly, a solution of each enzyme was prepared according to the manufacturer’s protocol in assay buffer. Separately, solutions of test compounds necessary to generate a seven-point dose response curve were prepared in half-log serial dilution. Test compounds were added to the plate, and the mixture was preincubated for 30 min at room temperature. A blank well (no enzyme) was included to assess the background signal, while the known inhibitors GC376, FF-FMK and B27-WT were used as positive controls for SARS-CoV-2 Mpro/3CLpro, cathepsin L, and calpain, respectively. The plates of SARS-CoV-2 Mpro, cathepsin L, and calpain were incubated with each fluorogenic substrate for 4, 0.3, and 1 h, respectively, at room temperature. Then fluorescence intensity was measured in a Cytation 5 cell imaging multi-mode reader (BioTek, Winooski, VT, USA)(excitation/emission: 360/460 nm, excitation/emission: 400/505 nm, and excitation/emission: 490/520 nm), respectively. End point fluorescence intensities were measured, and the blank value were subtracted from all values.
Higashi-Kuwata N., Tsuji K., Hayashi H., Bulut H., Kiso M., Imai M., Ogata-Aoki H., Ishii T., Kobayakawa T., Nakano K., Takamune N., Kishimoto N., Hattori S.I., Das D., Uemura Y., Shimizu Y., Aoki M., Hasegawa K., Suzuki S., Nishiyama A., Saruwatari J., Shimizu Y., Sukenaga Y., Takamatsu Y., Tsuchiya K., Maeda K., Yoshimura K., Iida S., Ozono S., Suzuki T., Okamura T., Misumi S., Kawaoka Y., Tamamura H, & Mitsuya H. (2023). Identification of SARS-CoV-2 Mpro inhibitors containing P1’ 4-fluorobenzothiazole moiety highly active against SARS-CoV-2. Nature Communications, 14, 1076.
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Publication 2023
Biological Assay Buffers Calpain Cathepsin L Cells Cysteine Proteases Enzyme Inhibitors Enzymes Fluorescence Fluorogenic Substrate GC376 Homo sapiens inhibitors SARS-CoV-2 Technique, Dilution

Top products related to «Calpain»

1
Calpain activity assay kit by Abcam
Sourced in United States, United Kingdom
The Calpain Activity Assay Kit is a laboratory tool used to measure the activity of calpain, a calcium-dependent cysteine protease, in biological samples. The kit provides the necessary reagents and protocols to quantify calpain activity through a colorimetric or fluorometric assay.
2
Calpain glo protease assay by Promega
Sourced in United States
The Calpain-Glo Protease Assay is a luminescent-based assay system designed to measure the activity of calpain proteases. It utilizes a proluminescent calpain substrate to provide a rapid, sensitive, and homogeneous method for detecting calpain activity in a variety of sample types.
3
Calpain activity fluorometric assay kit by Abcam
Sourced in United States
The Calpain Activity Fluorometric Assay Kit is a laboratory tool used to measure the activity of calpain enzymes. It utilizes a fluorogenic substrate to detect and quantify calpain activity in samples. The kit provides a simple and sensitive method for researchers to study calpain function in various biological systems.
4
Pvdf membrane by Merck Group
Sourced in United States, Germany, China, United Kingdom, Morocco, Ireland, France, Italy, Japan, Canada, Spain, Switzerland, New Zealand, India, Hong Kong, Sao Tome and Principe, Sweden, Netherlands, Australia, Belgium, Austria
PVDF membranes are a type of laboratory equipment used for a variety of applications. They are made from polyvinylidene fluoride (PVDF), a durable and chemically resistant material. PVDF membranes are known for their high mechanical strength, thermal stability, and resistance to a wide range of chemicals. They are commonly used in various filtration, separation, and analysis processes in scientific and research settings.
5
Ab65308 by Abcam
Sourced in United States
Ab65308 is a laboratory equipment product from Abcam. It is a tool designed for scientific research purposes.
6
Fetal bovine serum (fbs) by Thermo Fisher Scientific
Sourced in United States, China, United Kingdom, Germany, Australia, Japan, Canada, Italy, France, Switzerland, New Zealand, Brazil, Belgium, India, Spain, Israel, Austria, Poland, Ireland, Sweden, Macao, Netherlands, Denmark, Cameroon, Singapore, Portugal, Argentina, Holy See (Vatican City State), Morocco, Uruguay, Mexico, Thailand, Sao Tome and Principe, Hungary, Panama, Hong Kong, Norway, United Arab Emirates, Czechia, Russian Federation, Chile, Moldova, Republic of, Gabon, Palestine, State of, Saudi Arabia, Senegal
Fetal Bovine Serum (FBS) is a cell culture supplement derived from the blood of bovine fetuses. FBS provides a source of proteins, growth factors, and other components that support the growth and maintenance of various cell types in in vitro cell culture applications.
7
Calpeptin by Merck Group
Sourced in United States, Germany
Calpeptin is a laboratory reagent produced by Merck Group. It functions as a potent and selective inhibitor of calpains, a family of calcium-dependent cysteine proteases. Calpains play a role in various cellular processes. Calpeptin is commonly used in research applications to study the involvement of calpains in biological systems.
8
β actin by Merck Group
Sourced in United States, United Kingdom, Germany, China, Canada, Japan, Macao, Italy, Sao Tome and Principe, Israel, Spain, Denmark, France, Finland, Australia, Morocco, Ireland, Czechia, Sweden, Uruguay, Switzerland, Netherlands, Senegal
β-actin is a protein that is found in all eukaryotic cells and is involved in the structure and function of the cytoskeleton. It is a key component of the actin filaments that make up the cytoskeleton and plays a critical role in cell motility, cell division, and other cellular processes.
9
Calpain 1 by Abcam
Sourced in United States
Calpain-1 is a calcium-dependent intracellular cysteine protease. It plays a role in cellular processes such as signal transduction, cell motility, and apoptosis. Calpain-1 is involved in the cleavage of various cellular substrates.
10
Protease inhibitor cocktail by Merck Group
Sourced in United States, Germany, China, United Kingdom, Italy, Japan, Sao Tome and Principe, France, Canada, Macao, Switzerland, Spain, Australia, Israel, Hungary, Ireland, Denmark, Brazil, Poland, India, Mexico, Senegal, Netherlands, Singapore
The Protease Inhibitor Cocktail is a laboratory product designed to inhibit the activity of proteases, which are enzymes that can degrade proteins. It is a combination of various chemical compounds that work to prevent the breakdown of proteins in biological samples, allowing for more accurate analysis and preservation of protein integrity.

More about "Calpain"

Calpains are a family of calcium-activated, non-lysosomal cysteine proteases that play crucial roles in a variety of cellular processes, including cell signaling, cytoskeletal remodeling, and apoptosis.
These proteases have been implicated in the pathogenesis of numerous diseases, such as neurodegeneration, muscular dystrophy, and cancer.
Researching optimal experimental protocols for studying calpain activity and regulation is crucial for advancing our understanding of this important protease family and developing potential therapeutics.
Calpain-Glo Protease Assay and Calpain Activity Fluorometric Assay Kit are commonly used to measure calpain activity, while PVDF membranes and Ab65308 antibody can be utilized for western blot analysis of calpain expression.
Proper experimental design, with the use of appropriate controls (e.g., β-actin, Calpeptin) and protease inhibitor cocktails, is essential for ensuring the reproducibility and reliability of calpain research.
By leveraging PubCompare.ai's AI-powered technology, researchers can quickly identify the most effective and reproducible calpain research protocols from the literature, preprints, and patents, accelerating their investigations and contributing to the development of potential calpain-targeted therapies.