Protocols and Applications Guide (US Letter Size) - Promega

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||||||| 7Cell Signaling This study evaluated the feasibility of using the µARCS technology for nucleic acid polymerization assays. To ensure the efficient capture of the nucleic acid polymerization reaction and to minimize the nonspecific binding, the authors used a SAM2® Biotin Capture Membrane in the assay. In both studies, the nucleic acid substrate was biotinylated on one end and was bound to the SAM2® Membrane. PubMed Number: 12857381 Huynk, Q.K. et al. (2000) Characterization of the recombinant IKK1/IKK2 heterodimer. Mechanisms regulating kinase activity. J. Biol. Chem. 275, 25883–91. Kinase activity of IKK1/IKK2 was measured using a biotinylated IKBα peptide. The reaction was run and added to a SAM2® 96-well Biotin Capture plate. The plate was washed, dried, and γ33ATP was measured to indicate kinase activity. PubMed Number: 10823818 B. SignaTECT® Protein Kinase Assay Systems The SignaTECT® Protein Kinase Assay Systems use biotinylated peptide substrates in conjunction with the streptavidin-coated SAM2® Biotin Capture Membrane. The binding of biotin to the streptavidin is rapid and strong, and the association is unaffected by rigorous washing procedures, denaturing agents, wide extremes in pH, temperature and salt concentration. High signal-to-noise ratios are generated even with complex samples, while the high substrate capacity allows optimum reaction kinetics. The systems can be used to measure protein kinase activities using low femtomole levels of purified enzyme or crude cellular extracts. SignaTECT® Assays are available to measure protein tyrosine kinases (Cat.# V6480), cdc2 kinase (Cat.# V6430), cAMP-dependent protein kinase (Cat.# V7480), protein kinase C (Cat.# V7470), DNA-dependent protein kinase (Cat.# V7870) and calmodulin-dependent protein kinase (Cat.# V8161). As outlined in Figure 7.10, the assay steps and analysis of results are straightforward and require only common laboratory equipment. Following phosphorylation and binding of the biotinylated substrate to the numbered and partially cut squares of SAM2® Biotin Capture Membrane, unincorporated [γ-32P]ATP is removed by a simple washing procedure. This procedure also removes nonbiotinylated proteins that have been phosphorylated by other kinases in the sample. The bound, labeled substrate is then quantitated by scintillation counting or PhosphorImager® analysis. Typical results generated using the SignaTECT® Assays are presented in Figure 7.11. Protocols & Applications Guide www.promega.com rev. 3/09 Biotin–(C) 6 –XXXX(S/T/Y)XXXX Biotinylated Peptide Substrate + Protein Kinase Sample + Reaction Buffer + [γ−32P]ATP SAM 2® Biotin Capture Membrane Reaction Components PO 4 Combine peptide−substrate, [γ− 32 P]ATP, buffer and sample. Incubate at 30C. Terminate the kinase reaction with Termination Buffer. Biotin–(C) –XXXX(S/T/Y)XXXX 6 Capture the biotinylated peptide substrate by spotting reactions on individual squares on the capture membrane. PO4 Biotin–(C) –XXXX(S/T/Y)XXXX 6 Wash membrane to remove unbound reaction components. Quantitate Scintillation counter Phosphorimaging system Autoradiography Figure 7.10. The SignaTECT® Protein Kinase Assay protocol. PROTOCOLS & APPLICATIONS GUIDE 7-9
||||||| 7Cell Signaling CPM per Square (x 10 -3 ) 25 20 15 10 5 0 (+) PTK Biotinylated Peptide Substrate #1 (+) PTK Biotinylated Peptide Substrate #2 0 200 400 600 800 1,000 EGFR (fmol) 2.4 1.6 0.8 0 0 40 80 120 Figure 7.11. Linear detection of EGFR kinase activity with the SignaTECT® PTK Assay System. EGFR (Cat.# V5551) activity was measured in the presence of PTK Biotinylated Peptide Substrate 1 or PTK Biotinylated Peptide Substrate 2, provided with the SignaTECT® PTK System (Cat.# V6480). Inset: enlargement of the data using 120fmol of EGFR. Additional Resources for the SignaTect® Kinase Assay Systems Technical Bulletins and Manuals TB211 SignaTECT® Protein Tyrosine Kinase (PTK) Assay System Technical Bulletin (www.promega.com/tbs/tb211/tb211.html) TB227 SignaTECT® cdc2 Protein Kinase Assay System Technical Bulletin (www.promega.com/tbs/tb227/tb227.html) TB241 SignaTECT® cAMP-Dependent Protein Kinase Assay System Technical Bulletin (www.promega.com/tbs/tb241/tb241.html) TB242 SignaTECT® Protein Kinase C (PKC) Assay System Technical Bulletin (www.promega.com/tbs/tb242/tb242.html) TB250 SignaTECT® DNA-Dependent Protein Kinase Assay System Technical Bulletin (www.promega.com/tbs/tb250/tb250.html) TB279 SignaTECT® Calcium/Calmodulin-Dependent Protein Kinase (CaM KII) Assay System Technical Bulletin (www.promega.com/tbs/tb279/tb279.html) Promega Publications CN001 Store operated calcium entry activates at the GVBD stage of Xenopus meiosis (www.promega.com /cnotes/cn001/cn001_12.htm) Protocols & Applications Guide www.promega.com rev. 3/09 PN058 SAM2® Biotin Capture Membrane and SignaTECT® Protein Kinase Assay Systems (www.promega.com /pnotes/58/5189d/5189d.html) PN059 Detection and quantitation of protein tyrosine kinases (www.promega.com /pnotes/59/5644a/5644a.html) PN063 SignaTECT® DNA-Dependent Protein Kinase Assay System (www.promega.com /pnotes/63/8581_07/promega.html) PN076 Tools to study the activation of CaM KII in neuronal functions (www.promega.com /pnotes/76/8840_06/8840_06.html) BR095 Signal Transduction Resource (www.promega.com /guides/sigtrans_guide/default.htm) Citations Zhang, L. et al. (2004) A transforming growth factor beta-induced Smad3/Smad4 complex directly activates protein kinase A. Mol. Cel Biol. 24, 2169–80. These authors investigated the possible interaction between TGFβ and PKA signaling pathways using the SignaTECT® cAMP-Dependent Protein Kinase (PKA) Assay System. PubMed Number: 14966294 C. Other Kinase Assay Formats (non-radioactive) The PepTag® Protein Kinase Assays are fast and quantitative non-radioactive alternatives to [γ-32P]ATP-based assays for measuring protein kinase C (Cat.# V5330) and cAMP-dependent protein kinase (Cat.# V5340) activity. The assays use fluorescently-tagged peptide substrates with a net positive charge. Phosphorylation changes the charge of the peptide to a net negative, which influences the migration of the peptide in an agarose gel. This is the basis for detecting changes in phosphorylation via a rapid, 15-minute agarose gel separation (Figure 7.12). General PepTag® Assay Protocol Materials Required: • PepTag® Non-Radioactive PKC Assay (Cat.# V5330) or PepTag® Non-Radioactive cAMP-Dependent Protein Kinase Assay (Cat.# V5340) and protocol (#TB132 (www.promega.com/tbs/tb132/tb132.html)) • PKA or PKC dilution buffer • horizontal agarose gel apparatus • glycerol, 80% • Tris-HCl, 50mM (pH 8.0) • agarose, 0.8% in 50mM Tris-HCl (pH 8.0) • probe sonicator PROTOCOLS & APPLICATIONS GUIDE 7-10
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CONTENTS I. Nucleic Acid Amplificat
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| 1Nucleic Acid Amplification CONTE
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| 1Nucleic Acid Amplification Unamp
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| 1Nucleic Acid Amplification Capoz
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| 1Nucleic Acid Amplification is co
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| 1Nucleic Acid Amplification One i
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| 1Nucleic Acid Amplification React
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| 1Nucleic Acid Amplification comig
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| 1Nucleic Acid Amplification inclu
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| 1Nucleic Acid Amplification polym
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| 1Nucleic Acid Amplification Addit
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| 1Nucleic Acid Amplification 3. Pl
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| 1Nucleic Acid Amplification 13. S
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| 1Nucleic Acid Amplification IX. R
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| 1Nucleic Acid Amplification Hoppe
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|| 2RNA Interference CONTENTS I. RN
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|| 2RNA Interference zebrafish (War
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|| 2RNA Interference Additional Res
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|| 2RNA Interference 2. Combine sep
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|| 2RNA Interference Target Site Se
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|| 2RNA Interference Transient Tran
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|| 2RNA Interference VII. Reference
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|| 2RNA Interference Wianny, F. and
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||| 3Apoptosis I. Introduction A. C
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||| 3Apoptosis ER stress pathway (H
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||| 3Apoptosis pathways and demonst
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||| 3Apoptosis Qi, H. et al. (2003)
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|||| 4Cell Viability CONTENTS I. In
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|||| 4Cell Viability Table 4.1. Com
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|||| 4Cell Viability E. Homogeneous
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|||| 4Cell Viability Online Tools C
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|||||||||| 10Cell Imaging I. Introd
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||||||||||| 11Protein Purification
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||||||||||||| 13Cloning CONTENTS I.
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