Renaturation of Calcium/Calmodulin-Dependent Protein Kinase Activity after Electrophoretic Transfer from Sodium Dodecyl Sulfate-Polyacrylamide Gels to Membranes
A method is described for analyzing calcium/calmodulin-dependent protein kinase activity in crude or purified samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to PVDF membrane. The blotted protein is denatured in situ with guanidine H...
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| Veröffentlicht in: | Analytical biochemistry 1993-05, Vol.211 (1), p.131-138 |
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| creator | Shackelford, D.A. Zivin, J.A. |
| description | A method is described for analyzing calcium/calmodulin-dependent protein kinase activity in crude or purified samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to PVDF membrane. The blotted protein is denatured
in situ with guanidine HCl and renatured in buffer containing NP-40. The membrane with bound protein is incubated with [γ-
32P]ATP and buffer containing the activators Ca
2+ and calmodulin resulting in autophosphorylation of a subset of bound kinases. Two of the three major kinase activities detected in as little as 5 μg of crude brain or spinal cord homogenates are the α (
M
r = 50-52,000) and β (
M
r = 58-62,000) iso-forms of Ca
2+/calmodulin-dependent protein kinase II. A third unidentified kinase of
M
r = 90-95,000 is not dependent on Ca
2+ and calmodulin for activity. The membrane can be used for immunoblotting, phosphoamino acid analysis, or peptide mapping after the
in situ renaturation and phosphorylation procedure. Detection of kinase activity in this assay is dependent on autophosphorylation of the enzyme. Therefore another procedure is described in which the blotted proteins are denatured and renatured
in situ and assayed by measuring incorporation of phosphate into an exogenous peptide substrate specific for calcium/calmodulin-dependent protein kinase II. |
| doi_str_mv | 10.1006/abio.1993.1243 |
| format | Article |
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in situ with guanidine HCl and renatured in buffer containing NP-40. The membrane with bound protein is incubated with [γ-
32P]ATP and buffer containing the activators Ca
2+ and calmodulin resulting in autophosphorylation of a subset of bound kinases. Two of the three major kinase activities detected in as little as 5 μg of crude brain or spinal cord homogenates are the α (
M
r = 50-52,000) and β (
M
r = 58-62,000) iso-forms of Ca
2+/calmodulin-dependent protein kinase II. A third unidentified kinase of
M
r = 90-95,000 is not dependent on Ca
2+ and calmodulin for activity. The membrane can be used for immunoblotting, phosphoamino acid analysis, or peptide mapping after the
in situ renaturation and phosphorylation procedure. Detection of kinase activity in this assay is dependent on autophosphorylation of the enzyme. Therefore another procedure is described in which the blotted proteins are denatured and renatured
in situ and assayed by measuring incorporation of phosphate into an exogenous peptide substrate specific for calcium/calmodulin-dependent protein kinase II.</description><identifier>ISSN: 0003-2697</identifier><identifier>EISSN: 1096-0309</identifier><identifier>DOI: 10.1006/abio.1993.1243</identifier><identifier>PMID: 8391760</identifier><identifier>CODEN: ANBCA2</identifier><language>eng</language><publisher>San Diego, CA: Elsevier Inc</publisher><subject>Amino Acid Sequence ; Analytical, structural and metabolic biochemistry ; Animals ; Biological and medical sciences ; Brain - enzymology ; Calcium-Calmodulin-Dependent Protein Kinases ; Electrophoresis, Polyacrylamide Gel ; Enzymes and enzyme inhibitors ; Fundamental and applied biological sciences. Psychology ; Gels ; Isoenzymes - analysis ; Membranes, Artificial ; Molecular Sequence Data ; Peptide Fragments - analysis ; Phosphorylation ; Protein Denaturation ; Protein Kinases - chemistry ; Rabbits ; Rats ; Spinal Cord - enzymology ; Subcellular Fractions - enzymology ; Substrate Specificity ; Transferases</subject><ispartof>Analytical biochemistry, 1993-05, Vol.211 (1), p.131-138</ispartof><rights>1993 Academic Press</rights><rights>1993 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c434t-ef31ad6694d9e20bc0b2b506dd65b23eef59e1d6a29f08d4926508f5224afccd3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1006/abio.1993.1243$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3548,27923,27924,45994</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=4814080$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8391760$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Shackelford, D.A.</creatorcontrib><creatorcontrib>Zivin, J.A.</creatorcontrib><title>Renaturation of Calcium/Calmodulin-Dependent Protein Kinase Activity after Electrophoretic Transfer from Sodium Dodecyl Sulfate-Polyacrylamide Gels to Membranes</title><title>Analytical biochemistry</title><addtitle>Anal Biochem</addtitle><description>A method is described for analyzing calcium/calmodulin-dependent protein kinase activity in crude or purified samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to PVDF membrane. The blotted protein is denatured
in situ with guanidine HCl and renatured in buffer containing NP-40. The membrane with bound protein is incubated with [γ-
32P]ATP and buffer containing the activators Ca
2+ and calmodulin resulting in autophosphorylation of a subset of bound kinases. Two of the three major kinase activities detected in as little as 5 μg of crude brain or spinal cord homogenates are the α (
M
r = 50-52,000) and β (
M
r = 58-62,000) iso-forms of Ca
2+/calmodulin-dependent protein kinase II. A third unidentified kinase of
M
r = 90-95,000 is not dependent on Ca
2+ and calmodulin for activity. The membrane can be used for immunoblotting, phosphoamino acid analysis, or peptide mapping after the
in situ renaturation and phosphorylation procedure. Detection of kinase activity in this assay is dependent on autophosphorylation of the enzyme. Therefore another procedure is described in which the blotted proteins are denatured and renatured
in situ and assayed by measuring incorporation of phosphate into an exogenous peptide substrate specific for calcium/calmodulin-dependent protein kinase II.</description><subject>Amino Acid Sequence</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>Brain - enzymology</subject><subject>Calcium-Calmodulin-Dependent Protein Kinases</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gels</subject><subject>Isoenzymes - analysis</subject><subject>Membranes, Artificial</subject><subject>Molecular Sequence Data</subject><subject>Peptide Fragments - analysis</subject><subject>Phosphorylation</subject><subject>Protein Denaturation</subject><subject>Protein Kinases - chemistry</subject><subject>Rabbits</subject><subject>Rats</subject><subject>Spinal Cord - enzymology</subject><subject>Subcellular Fractions - enzymology</subject><subject>Substrate Specificity</subject><subject>Transferases</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1993</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kU9v1DAQxS0EKkvhyg3JB9Rbtv6TeONjtS0FUURFy9ly7LEwcuLFdirl2_BRcbSr3jjN4f3maeY9hN5TsqWEiEs9-LilUvItZS1_gTaUSNEQTuRLtCGE8IYJuXuN3uT8mxBK206cobOeS7oTZIP-_oBJlznp4uOEo8N7HYyfx8s6x2jn4KfmGg4wWZgKvk-xgJ_wVz_pDPjKFP_ky4K1K5DwTQBTUjz8igmKN_gx6Sm7KrgUR_wQbfXF19GCWQJ-mIPTBZr7GBZt0hL06C3gWwgZl4i_wTjUdchv0SunQ4Z3p3mOfn66edx_bu6-337ZX901puVtacBxqq0QsrUSGBkMGdjQEWGt6AbGAVwngVqhmXSkt61koiO96xhrtTPG8nN0cfQ9pPhnhlzU6LOBEOoRcc5q1_W8ZTtSwe0RNCnmnMCpQ_KjTouiRK2VqLUStVai1krqwoeT8zyMYJ_xUwdV_3jSdTY6uPq28fkZa3vakn7F-iNWE4InD0ll42EyYH2quSsb_f8u-AcYTaul</recordid><startdate>19930515</startdate><enddate>19930515</enddate><creator>Shackelford, D.A.</creator><creator>Zivin, J.A.</creator><general>Elsevier Inc</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>19930515</creationdate><title>Renaturation of Calcium/Calmodulin-Dependent Protein Kinase Activity after Electrophoretic Transfer from Sodium Dodecyl Sulfate-Polyacrylamide Gels to Membranes</title><author>Shackelford, D.A. ; Zivin, J.A.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c434t-ef31ad6694d9e20bc0b2b506dd65b23eef59e1d6a29f08d4926508f5224afccd3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1993</creationdate><topic>Amino Acid Sequence</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>Brain - enzymology</topic><topic>Calcium-Calmodulin-Dependent Protein Kinases</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gels</topic><topic>Isoenzymes - analysis</topic><topic>Membranes, Artificial</topic><topic>Molecular Sequence Data</topic><topic>Peptide Fragments - analysis</topic><topic>Phosphorylation</topic><topic>Protein Denaturation</topic><topic>Protein Kinases - chemistry</topic><topic>Rabbits</topic><topic>Rats</topic><topic>Spinal Cord - enzymology</topic><topic>Subcellular Fractions - enzymology</topic><topic>Substrate Specificity</topic><topic>Transferases</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Shackelford, D.A.</creatorcontrib><creatorcontrib>Zivin, J.A.</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Shackelford, D.A.</au><au>Zivin, J.A.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Renaturation of Calcium/Calmodulin-Dependent Protein Kinase Activity after Electrophoretic Transfer from Sodium Dodecyl Sulfate-Polyacrylamide Gels to Membranes</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>1993-05-15</date><risdate>1993</risdate><volume>211</volume><issue>1</issue><spage>131</spage><epage>138</epage><pages>131-138</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><coden>ANBCA2</coden><abstract>A method is described for analyzing calcium/calmodulin-dependent protein kinase activity in crude or purified samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to PVDF membrane. The blotted protein is denatured
in situ with guanidine HCl and renatured in buffer containing NP-40. The membrane with bound protein is incubated with [γ-
32P]ATP and buffer containing the activators Ca
2+ and calmodulin resulting in autophosphorylation of a subset of bound kinases. Two of the three major kinase activities detected in as little as 5 μg of crude brain or spinal cord homogenates are the α (
M
r = 50-52,000) and β (
M
r = 58-62,000) iso-forms of Ca
2+/calmodulin-dependent protein kinase II. A third unidentified kinase of
M
r = 90-95,000 is not dependent on Ca
2+ and calmodulin for activity. The membrane can be used for immunoblotting, phosphoamino acid analysis, or peptide mapping after the
in situ renaturation and phosphorylation procedure. Detection of kinase activity in this assay is dependent on autophosphorylation of the enzyme. Therefore another procedure is described in which the blotted proteins are denatured and renatured
in situ and assayed by measuring incorporation of phosphate into an exogenous peptide substrate specific for calcium/calmodulin-dependent protein kinase II.</abstract><cop>San Diego, CA</cop><pub>Elsevier Inc</pub><pmid>8391760</pmid><doi>10.1006/abio.1993.1243</doi><tpages>8</tpages></addata></record> |
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| ispartof | Analytical biochemistry, 1993-05, Vol.211 (1), p.131-138 |
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| language | eng |
| recordid | cdi_proquest_miscellaneous_75834270 |
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| subjects | Amino Acid Sequence Analytical, structural and metabolic biochemistry Animals Biological and medical sciences Brain - enzymology Calcium-Calmodulin-Dependent Protein Kinases Electrophoresis, Polyacrylamide Gel Enzymes and enzyme inhibitors Fundamental and applied biological sciences. Psychology Gels Isoenzymes - analysis Membranes, Artificial Molecular Sequence Data Peptide Fragments - analysis Phosphorylation Protein Denaturation Protein Kinases - chemistry Rabbits Rats Spinal Cord - enzymology Subcellular Fractions - enzymology Substrate Specificity Transferases |
| title | Renaturation of Calcium/Calmodulin-Dependent Protein Kinase Activity after Electrophoretic Transfer from Sodium Dodecyl Sulfate-Polyacrylamide Gels to Membranes |
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