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|Baculovirus-Insect Cell lysate that Human CAMK1G / CLICK III / CaMKIgamma transfected / overexpressed for Western blot (WB) positive control. The whole cell lysate is provided in 1X Sample Buffer (1X modified RIPA buffer+1X SDS loading buffer).|
|A DNA sequence encoding the human CAMK1G isoform 1 (Q96NX5-1) (Met 1-Met 476) was fused with the N-terminal polyhistidine-tagged GST tag at the N-terminus.|
|The recombinant human CAMK1G/GST chimera consists of 713 amino acids and has a calculated molecular mass of 81 kDa. It migrates as an approximately 75 kDa band in SDS-PAGE under reducing conditions.|
|Cell lysate was prepared by homogenization in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.|
|Modified RIPA Lysis Buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 1mM PMSF.|
|12.5% SDS-PAGE Stained with Coomassie Blue after protein purification.|
|Samples are stable for up to twelve months from date of receipt.|
|1. Centrifuge the tube for a few seconds and ensure the pellet at the bottom of the tube. 2. Re-dissolve the pellet using 200μL pure water and boil for 2-5 min. 3. Store the lyophilized cell lysate at 4℃. After re-dissolution, recommend to aliquot it into smaller quantities and store at -80℃.|
|1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).|
|Store at 4℃. After re-dissolution, aliquot and store at -80℃.|
|Western blot (WB): Use at an assay dependent dilution.|
Other Applications: Not tested.
Optimal dilutions/concentrations should be determined by the end user.
Calmodulin-Dependent Protein Kinase (CaM Kinase) is a kind of protein phosphorylate multiple downstream targets. Concentration of cytosolic calcium functions as a second messenger that mediates a wide range of cellular responses. Calcium binds to calcium binding proteins (calmodulin/CaM) and stimulates the activity of a variety of enzymes, including CaM kinases referred to as CaM-kinases (CaMKs), such as CaMKI, CaMKII, CaMKIV and CaMKK. Calmodulin-dependent protein kinase CL3/CaMKIγ is a memberane-anchored CaMK belonging to the CaM kinase family. Its C-terminal region is uniquely modified by two sequential lipidification steps: prenylation followed by a kinase-activity-regulated palmitoylation. These modifications are essential for CaMKIγ membrane anchoring and targeting into detergent-resistant lipid microdomains in the dendrites. It has been found that CaMKIγ critically contributed to BDNF-stimulated dendritic growth. Raft insertion of CaMKIγ specifically promoted dendritogenesis of cortical neurons by acting upstream of RacGEF STEF and Rac, both present in lipid rafts. Thus, CaMKIγ may represent a key element in the Ca2+-dependent and lipid-raft-delineated switch that turns on extrinsic activity-regulated dendrite formation in developing cortical neurons.