ATP, calmodulin, calmodulin-agarose, protease inhibitor cocktail, anti-α-CaMKII antibody, secondary antibody conjugates, PMSF (phenylmethylsulfonylfluoride) and DTT (dithiothreitol) were from Sigma Chemicals, USA. Phosphocellulose was from Whatman, UK. IPTG (Isopropylthiogalactoside) and glutathione sepharose 4B were from GE, USA. Pierce glutathione-agarose was from Thermo Fisher Scientific. Phospho-Thr286-α-CaMKII antibody, was either from Sigma-Aldrich or from Cell Signaling Technology. Oligonucleotides were obtained from SigmaGenosys, USA. Quikchange site directed mutagenesis kit was from Stratagene, USA. Nitrocellulose paper was from PALL Gelmann. [γ-32P]ATP was from Bhabha Atomic Research Centre, India. Anti-glutathione-S-transferase (GST) antibody was from Santacruz Biotechnology Inc., USA. Protein phosphatase 1 (PP1) was from New England Biolabs, USA. GST-CaMKIINα plasmid was a gift from Dr. P. Rangarajan, Department of Biochemistry, Indian Institute of Science, Bangalore, India.
Calmodulin agarose
Manufactured by Merck Group
Sourced in United States, Germany
Calmodulin-agarose is a chromatography medium used for the purification of calmodulin-binding proteins. It consists of calmodulin covalently coupled to a cross-linked agarose resin. This medium can be used to capture and isolate various calmodulin-dependent proteins from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Protein phosphatase 1, by New England Biolabs (1 mentions)
Calmodulin, by Merck Group (1 mentions)
Anti glutathione s transferase antibody, by Santa Cruz Biotechnology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Pierce glutathione agarose, by Thermo Fisher Scientific (1 mentions)
Whatman, by Cytiva (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Dextrose, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Phosphatidylserine, by Merck Group (1 mentions)
Methylene blue, by Merck Group (1 mentions)
4 protocols using calmodulin agarose
1
Purification and Characterization of CaMKII
2
Purification of FtsQ-RNC Constructs
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We purified FtsQ-RNC constructs as previously described [12 (link)]. These RNCs contained nascent chain based on 101 N-terminal residues of FtsQ. This RNC contained in addition to the His tag on the ribosome a calmodulin binding peptide for an additional affinity purification step. The specific stalling sequence motif SecM contained the FXXXXWIXXXXGIRAGP motif. Nascent chains were expressed for 2 h in JE28 cells containing his-tagged ribosomes [17 (link)] by addition of 2 g/L arabinose. Pelleted cells were lysed in 20 mM Tris (pH 7.6), 10 mM MgCl2, 150 mM KCl, and 30 mM NH4Cl in a homogenizer at low pressure (15–17 kPsi). Two subsequent affinity purifications with Ni-NTAagarose (Quiagen, Hilden, Germany) and calmodulin agarose (Sigma-Aldrich, St. Louis, MO, USA) served to first selectively purify ribosomes and second to select those ribosomes that contained a stalled nascent chain.
3
Yeast Synthetic Drop-out Media Preparation
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Yeast synthetic drop-out media supplement without leucine, yeast nitrogen base without amino acids, dextrose, polyoxyethylene 10 lauryl ether (C12E10), L -α-phosphatidylcholine type XVI-E (Sigma) from fresh egg yolk, brain extract (BE) Type I Folch Fraction I from bovine brain containing approximately (w/w) 10% PI, 50% PS, and other lipids, phosphodiesterase 3′, 5′ cyclic nucleotide activator (calmodulin) from bovine brain, calmodulin-agarose, ATP (disodium salt, vanadium-free), sodium dodecyl sulfate (SDS), and all other chemicals were obtained from Sigma.
4
Tau Aggregation Inhibition Assay
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Phosphatidylcholine (PC) type XI-E from egg yolk, phosphatidylserine (PS), calmodulin-agarose, and methylene blue (MB) were obtained from Sigma. methylene blue was prepared as stock solution of 5 mg/mL (w/v) and made freshly for each separate experiment. Tau 441 was obtained from Enzo Life Sciences. Pig brains were obtained from a local slaughterhouse and human autopsy brain tissues (medium frontal gyrus, a brain region highly affected in AD) were supplied by the Netherlands Brain Bank (NBB, Netherlands Institute for Neuroscience, Amsterdam, Netherlands) and obtained from 8 neuropathologically confirmed AD patients (age 79 ± 2yr, Braak stage V/VI) and from 7 age-matched non-diseased control subjects. Procedures, information, and consent forms of the NBB have been approved by the Medical Ethics Committee of the Vrije Universiteit Amsterdam Medical Centre on 30 April 2009. The project identification code is Avila(537). Investigations using human tissues were carried out following the rules of the Declaration of Helsinki of 1975 (https://www.wma.net/what-we-do/medical-ethics/declaration-of-helsinki/ ), revised in 2013.
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