Protocols and Applications Guide (US Letter Size) - Promega

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|||||||||| 10Cell Imaging Online Tools Apoptosis Assistant (www.promega.com/apoasst/) Antibody Assistant (www.promega.com /techserv/tools/abasst/) Citations Davidson, B. et al. (2003) Expression and activation of the nerve growth factor receptor TrkA in serous ovarian carcinoma. Clin. Cancer Res. 9, 2248–59. Anti-PARP p85 Fragment pAb was used to stain human peritoneal and pleural effusions. PubMed Number: 12796393 O'Brien, M.A., Moravec, R.A. and Riss, T. (2001) Poly (ADP-ribose) polymerase cleavage monitored in situ in apoptotic cells. Biotechniques. 30, 886–91. The authors demonstrate specificity of an affinity-purified polyclonal antibody to the p85 fragment of PARP with Western blots that show that the antibody recognizes the 85kDa (p85) fragment of PARP but not full-length PARP. PubMed Number: 11314271 B. Antibodies and Cellular Markers for Studying Cell Signaling Pathways Promega provides a variety of phosphorylation-specific antibodies for studying cell signaling pathways. These antibodies and example protocols are discussed in detail in the Protocols and Applications Guide chapter on Cell Signaling (www.promega.com/paguide/chap7.htm#title4). Brief descriptions of these products are provided below. Anti-ACTIVE® Phosphorylation-Specific Antibodies The Anti-ACTIVE® phosphorylation-specific antibodies were developed to provide an accurate measure of enzyme activation. These antibodies specifically recognize the active, phosphorylated form of a given kinase. The Anti-ACTIVE® Antibodies are raised against phosphorylated peptide sequences present in the activating loop of a number of protein kinases. Whether used in Western analysis, immunocytochemistry or immunohistochemical staining, the Anti-ACTIVE® MAPK, JNK, p38 and CaM KII Antibodies will recognize only the active form of the enzyme. Anti-ACTIVE® MAPK, pAb, Rabbit, (pTEpY) This antibody is an affinity-purified polyclonal antibody that specifically recognizes the dually phosphorylated, active form of MAPK. The antibody is raised against a dually phosphorylated peptide sequence representing the catalytic core of the active ERK enzyme and recognizes the active forms of ERK1, ERK2 and ERK7. Anti-ACTIVE® JNK pAb, Rabbit, (pTPpY) Anti-ACTIVE® JNK pAb is an affinity-purified polyclonal antibody that recognizes the dually phosphorylated, active form of cJun N-terminal protein Kinase (JNK). Anti-ACTIVE® JNK pAb is raised against a dually Protocols & Applications Guide www.promega.com rev. 3/09 phosphorylated peptide sequence representing the catalytic core of the active JNK enzyme. The antibody recognizes the active forms of JNK1, JNK2, and JNK3 isoforms. Anti-ACTIVE® p38 pAb, Rabbit, (pTGpY) Anti-ACTIVE® p38 Ab, Rabbit, is an affinity-purified polyclonal antibody that recognizes the active form of p38 kinase. The Anti-ACTIVE® p38 pAb is raised against the dually phosphorylated peptide sequence representing the catalytic core of the active p38 enzyme. The Anti-ACTIVE® p38 pAb recognizes the active forms of p38α, γ, and δ isoforms. Additional Resources for the Anti-ACTIVE® Antibodies Technical Bulletins and Manuals TB262 Anti-ACTIVE® MAPK, p38 and JNK Polyclonal Antibodies and Anti-ACTIVE® Qualified Secondary Antibody Conjugates (www.promega.com/tbs/tb262/tb262.html) Promega Publications PN069 New Anti-ACTIVE® MAPK and 'pan ERK 1/2' antibodies for Western analysis (www.promega.com /pnotes/69/7542_09/7542_09.html) PN076 Technically speaking: Anti-ACTIVE® Antibodies and MAPK signaling pathways (www.promega.com /pnotes/76/8840_23/8840_23.html) PN080 Demonstration of immunohistochemical staining using Promega's Anti-ACTIVE® and apoptosis Antibodies (www.promega.com /pnotes/80/9748_20/9748_20.html) Online Tools Antibody Assistant (www.promega.com /techserv/tools/abasst/) Citations Hsu, C.Y. et al. (2004) Characterization of active mitogen-activated protein kinase in ovarian serous carcinomas Clin. Can. Res. 10, 6432–6. The Anti-ACTIVE® MAPK polyclonal antibody was used to immunohistochemically stain and type patient ovarian serous carcinomas using paraffin-fixed tissue sections on tissue microarrays. Western blots were also performed on tissue lysates using a 1:3,000 dilution of the antibody. PubMed Number: 15475429 Le'Negrate, G. et al. (2003) Downregulation of caspases and Fas ligand expression, and increased lifespan of neutrophils after transmigration across intestinal epithelium Cell Death Differ. 10, 153–62. Anti-ACTIVE® JNK pAb was used in immunoblot analysis of human polymorphonuclear leukocyte protein lysates. PubMed Number: 12700643 Aballay, A. et al. (2003) Caenorhabditis elegans innate immune response triggered by Salmonella enterica requires intact LPS PROTOCOLS & APPLICATIONS GUIDE 10-14
|||||||||| 10Cell Imaging and is mediated by a MAPK signaling pathway Curr. Biol. 13, 47–52. Activation of the p38 homolog in the worm was monitored by Western analysis using the Anti-ACTIVE® p38 pAb. PubMed Number: 12526744 Phosphorylation-Specific CaM KII Antibody This antibody recognizes calcium/calmodulin-dependent protein kinase, CaM KII, that is phosphorylated on threonine 286. The Anti-ACTIVE® CaM KII pAb (Cat.# V1111) was raised against the phosphothreonine-containing peptide derived from this region. Additional Information for the Anti-ACTIVE® CaM KII pAb Technical Bulletins and Manuals TB264 Anti-ACTIVE® CaM KII pAb, (pT286) and Anti-ACTIVE® Qualified Secondary Antibody Conjugates Technical Bulletin (www.promega.com/tbs/tb264/tb264.html) Promega Publications PN067 Anti-ACTIVE® Antibody for specific detection of phosphorylated CaM KII protein kinase (www.promega.com /pnotes/67/7201_09/7201_09.html) BR095 Signal Transduction Resource (www.promega.com /guides/sigtrans_guide/default.htm) Online Tools Antibody Assistant (www.promega.com /techserv/tools/abasst/) Citations Matsumoto, Y. and Maller, J.L. (2002) Calcium, calmodulin and CaM KII requirement for initiation of centrosome duplication in Xenopus egg extracts Science 295, 499–502. CaM KII (281-309) was added to metaphase-arrested extracts. After adding calcium, the extracts were incubated at room temperature. Anti-ACTIVE® CaM KII pAb and Anti-ACTIVE® Qualified HRP secondary antibodies were used to probe immunoblots for phospho-T286 CaM KIIα. PubMed Number: 11799245 C. Marker Antibodies Anti-βIII Tubulin mAb Anti-βIII Tubulin mAb (Cat.# G7121) is a protein G-purified IgG1 monoclonal antibody (from clone 5G8) raised in mice against a peptide (EAQGPK) corresponding to the C-terminus of βIII tubulin. It is directed against βIII tubulin, a specific marker for neurons. The major use of this antibody is for labeling neurons in tissue sections and cell culture. The antibody performs in frozen and paraffin-embedded sections of rat brain, cerebellum and Protocols & Applications Guide www.promega.com rev. 3/09 spinal cord, human and rat fetal CNS progenitor cell cultures and adult human paraffin-embedded brain (Figure 10.12). Figure 10.12. Immunostaining for βIII tubulin in rat cerebellum using Anti-βIII Tubulin mAb. Paraffin-embedded rat brain section double-immunofluorescence- labeled with the primary antibody and detected using an anti-mouse Cy®3-conjugated secondary antibody (yellow-green). Nuclei were stained with DAPI (blue). Protocols developed and performed at Promega. • Immunogen: Peptide corresponding to the C-terminus (EAQGPK) of βIII tubulin. • Antibody Form: Mouse monoclonal IgG1 (clone 5G8), 1mg/ml in PBS containing no preservatives. • Specificity: Cross-reacts with most mammalian species. Does not label nonneuronal cells (e.g., astrocytes). • Suggested Working Dilutions: Immunocytochemistry (1:2,000), Immunohistochemistry (1:2,000), Western blotting (1:1,000 dilution). Additional Resources for β-III Tubulin mAb Promega Publications CN012 Perform multicolor live- and fixed-cell imaging applications with the HaloTag® Interchangeable Labeling Technology (www.promega.com /cnotes/cn012/cn012_04.htm) NN020 CellTiter-Glo® Luminescent Cell Viability Assay: Primary neurons and human neuroblastoma SH-SY5Y cells (www.promega.com /nnotes/nn020/20_05.htm) NN019 Rat neurospheres express mRNAs for TrkB, BDNF, NT-3 and p75 (www.promega.com /nnotes/nn019/19_18.htm) NN018 Specific labeling of neurons and glia in mixed cerebrocortical cultures (www.promega.com /nnotes/nn018/018_10.htm) NN018 Imaging with Promega Reagents: Anti-βIII Tubulin mAb (1,439kb PDF) (www.promega.com /nnotes/nn018/18_8.pdf) 2137TA PROTOCOLS & APPLICATIONS GUIDE 10-15
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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 Buffer Substrate Tha
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||| 3Apoptosis Qi, H. et al. (2003)
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||| 3Apoptosis Fluorescence (560/59
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||| 3Apoptosis Ellis, H.M. and Horv
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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 Crouch, S.P.M.
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||||| 5Protein Expression I. Introd
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|||||| 6Expression Analysis I. Intr
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||||||| 7Cell Signaling CONTENTS I.
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||||||| 7Cell Signaling SMALL GTP-B
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||||||| 7Cell Signaling HO S N N S
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||||||| 7Cell Signaling PN083 Intro
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||||||| 7Cell Signaling CPM per Squ
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||||||| 7Cell Signaling A. Nitrocel
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||||||| 7Cell Signaling (continued
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||||||| 7Cell Signaling Promega Pub
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||||||| 7Cell Signaling Nonphosphor
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|||||||| 8Bioluminescence Reporters
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Table 8.1. pGL4 Luciferase Reporter
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Page 271 and 272: ||||||||||||| 13Cloning CONTENTS I.
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||||||||||||| 13Cloning Promega Pub
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||||||||||||| 13Cloning Figure 13.9
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