I would like more information on the EF1 alpha promoter. Are there any references available?

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Here are some helpful references concerning the EF-1 alpha promoter:
---Uetsuki et al (1989) J. Biol. Chem. 264:5791 ---Kim et al (1990) Gene 91:217 ---Goldman et al (1996) BioTechniques 21:1013 ---Mizushima and Nagata (1990) Nucleic Acids Research 18:5322

Answer Id: E3469

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49e65879025172d4e640f989226b337a_FAQ

Is there a reasonable replacement for the NuPAGE® antioxidant for gel electrophoresis?

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Answer

One can use 0.002% thioglycolic acid in the upper buffer reservoir. This is a good scavenger of free radicals. The reference to this is described by Hunkapiller et al, Methods of Enzymology, (91), 399, 1983. Caution should be taken when using this method since this compound is both toxic and expensive. In addition, the TGA must be fresh as it tends to become oxidized itself over time. Oxidized TGA will actually promote sample re-oxidation.

Answer Id: E3571

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16138e6a50fd849825e377eca40d8390_FAQ

Do you have any references for isolation of vesicles and organelles using Dynabeads® magnetic beads?

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Here is a sampling of references for organelle isolation, including vesicles and exosomes.

Dynabeads® products used for exosome isolation via an antibody plus some other references where Dynabeads® M-500 Subcellular has been used to isolate other types of organelles:

Rabesandratana H et al. (1998) Decay-accelerating factor (CD55) and membrane inhibitor of reactive lysis (CD59) are released within exosomes during in vitro maturation of reticulocytes. Blood 91:2573-2580.

Allan BB et al. (2001)Rab1 recruitment of p115 into a cis-SNARE complex: programming budding COPII vesicles for fusion. Science 289:444-448.

Li X et al. (2001) Na+-H+ exchanger 3 (NHE3) is present in lipid rafts in the rabbit ileal brush border: a role for rafts in trafficking and rapid stimulation of NHE3. J Physiol 537:537-552.

Some references for isolation of endosomes and lysosomes.

Gagescu R (2000) The recycling of endosome of Madin-Darby canine kidney cells is a mildly acidic compartment rich in raft components. Mol Biol Cell 11:2775-2791.

Provoda CJ et al. (2000) Evidence for a primary endocytic vesicle involved in synaptic vesicle biogenesis. J Biol Chem 275:7004-7012.

Tjelle TE et al. (1998) Degradation of phagosomal components in late endocytic organelles. J Cell Sci 111:141-148.

Vitale N et al. (1998) Localization of ADP-ribosylation factor domain protein (ARD1) in lysosomes and Golgi apparatus. Proc Natl Acad Sci USA 95:8613-8618.

Clayton A et al. (2001) Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry. J Immunol Methods 247:163-174.

Marchand S et al. (2001) Differential targeting of components of the dystrophin complex to the postsynaptic membrane. Eur J Neurosci 13: 221-229.

Some references to articles where vesicles transporting from the ER have been isolated and related references:

Allan BB et al. (2000) Rab1 recruitment of p115 into a cis-SNARE complex: programming budding COPII vesicles for fusion. Science 289:444-448.

Seemann J et al. (2002) Partitioning of the matrix fraction of the Golgi apparatus during mitosis in animal cells. Science 295:848-851.

Jesch SA, Linstedt AD (19980 The Golgi and endoplasmic reticulum remain independent during mitosis and HeLa cells. Mol Biol Cell 9:623-635.

Sinai AP et al. (1997) Association of host cell endoplasmic reticulum and mitochondria with the Toxoplasma gondii parasitophorous vacuole membrane: a high affinity interaction. J Cell Sci 110:2117-2128.

Sans N et al. (2001) Synapse-associated protein 97 selectively associates with a subset of AMPA receptors early in their biosynthetic pathway. J Neurosci 21:7506-7516.

Abo-Hashema KAH et al. (1999) Evidence for triacylglycerol synthesis in the lumen of microsomes via a lipolysis-esterification pathway involving carnitine acyltransferases. J Biol Chem 274:35577-35582.

References where Dynabeads® magnetic beads have been used to isolate mitochondria and other organelles, or to isolate mitochondrial or chloroplast DNA using Dynabeads® magnetic beads:

Lüers GH et al. (1998) Immuno-isolation of highly purified peroxisomes using magnetic beads and continuous immunomagnetic sorting. Electrophoresis 19:1205-1210.

Sexton PS, Cenedella RJ. (2002) Immunomagnetic capture of lens membrane fractions containing steroid binding protein. Biochem Biophys Res Commun 295:1027-1031.

Hansen GH et al. (2001) Lipid rafts exist as stable cholesterol-independent microdomains in the brush border membrane of enterocytes. J Biol Chem 276:32338-32344.

Sinai AP et al. (1997) Association of host cell endoplasmic reticulum and mitochondria with the Toxoplasma gondii parasitophorous vacuole membrane: a high affinity interaction. J Cell Sci 110:2117-2128.

Jones SM et al. (1998) Immunoisolation of organelles using magnetic solid supports, p. 12-14 in Cell Biology: A Laboratory Handbook, 2nd edn., Academic Press.

Müsch A et al. (1997) Myosin II is involved in the production of constitutive transport vesicles from the TGN. J Cell Biol 138:291-306.

Herrnstadt C et al. (1999) A novel mitochondrial DNA-like sequence in the human nuclear genome. Genomics 60:67-77.

Kausch AP et al. (1999) Organelle isolation by magnetic immunoabsorption. BioTechniques 26 (2):336-343.

Hopwood AJ et al. (1996) DNA typing from human faeces. Int J Legal Med 108:237-243.

Marchington DR et al. (1997) Homopolymeric tract heteroplasmy in mtDNA from tissues and single oocytes: support for a genetic bottleneck. Am J Hum Genet 60:408-416.

Piercy R et al. (1993) The application of mitochondrial DNA typing to the study of white Caucasian genetic identification. Int J Legal Med 106:85-90.

Fangan BM et al. (1994) A general approach for PCR-amplification and sequencing of chloroplast DNA from crude vascular plant and algal tissue. BioTechniques 16:484-494.

Ferris C et al. (1995) Using chloroplast DNA to trace postglacial migration routes of oaks into Britain. Mol Ecol 4:731-738, 1995.

Answer Id: E6150

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7f368222a8779ef07bd0a7d88a0450d0_FAQ

What is the cause of smeary artifacts down the lanes of a Tricine gel and how can this be prevented?

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Answer

Protein samples are possibly reoxidizing before the run is complete in the Tricine gel system. Since Tricine is a glycine derivative, the running pH ranges of the two systems are different. As a consequence, reduced samples tend to oxidize more in the Tricine system. Adding more reducing agent will not solve the problem.

One option is to alkylate the sample by reducing with 20 mM DTT at 70°C for 30 min, followed by 50 mM iodoacetic acid to alkylate.

Another method which inhibits oxidation is the addition of thioglycolic acid (TGA) to the running buffer. The reference to this is described by Hunkapiller et al, Methods of Enzymology, (91), 399, 1983.

Caution should be taken when using this method since this compound is both toxic and expensive. In addition, the TGA must be fresh as it tends to become oxidized itself over time. Oxidized TGA will actually promote sample re-oxidation.

Answer Id: E3589

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7e4476c5d49f005dcdbb8e9c1ec9bb74_FAQ

Do you have any references for isolation of mice endothelial cells using Dynabeads® magnetic beads?

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Answer

These are several references describing isolation of endothelial cells from various murine samples or tissues using different endothelial markers. In addition, we have included a brief description of the Dynabeads® product used and the epithelial cell target antigen for each of the references.

Carilo A et al. (2004) Transcription regulation of TNF-alpha-early response genes by poly(ADP-ribose) polymerase-1 in murine heart endothelial cells. Nucleic Acids Res 32(2): 757-766.
Mouse heart endothelial cells were isolated using rat anti-mouse CD31 (clone MEC13.3, BD Pharmingen) and Dynabeads® M-450 sheep anti-rat IgG.

van Wijngaarden J et al. (2004) Identification of differentially expressed genes in a renal cell carcinoma tumor model after endostatin-treatment. Lab Invest 84:1472-1483.
Mouse endothelial cells were isolated using rat anti-mouse CD31 (clone ER-MP12) and Dynabeads® M-450 sheep anti-rat IgG.

Kataoka H et al. (2003) Protease-activated receptors 1 and 4 mediate thrombin signaling in endothelial cells. Blood 102(9):3224-3231.
Mouse endothelial cells were isolated using Dynabeads® M-450 sheep anti-rat IgG and a rat anti-mouse ICAM-2 (CD102) antibody from BD Pharmingen.

Lim YC et al. (2003) Heterogeneity of endothelial cells from different organ sites in T-cell subset recruitment. Am J Pathol 162(5):1591-1601.
Dynabeads® sheep anti-rat IgG were coated with either anti-PECAM-1 (CD31) or anti-ICAM-2 (CD102) monoclonal antibody (2.5 μg antibody for 2 x 10e7 beads) and used for isolation of mouse lung or heart endothelial cells.

Camerer E et al. (2002) Genetic evidence that protease-activated receptors mediate factor Xa signaling in endothelial cells. J Biol Chem 277(18):16081-16087.
Mouse ECs isolated using Dynabeads® magnetic beads coated with a rat anti-mouse ICAM-2 (CD102) antibody (BD Pharmingen). Purity was more than 90%.

Armstrong LC et al. (2002) Thrombospondin 2 inhibits microvascular endothelial cell proliferation by a caspase-independent mechanism. Mol Biol Cell 13(6):1893-1905.
Mouse lung epithelial cells were isolated using Dynabeads® sheep anti-rat IgG and a BD Pharmingen rat anti-mouse ICAM-2 (CD102) antibody.

Tiruppathi C et al. (2002) Impairment of store-operated Ca2+ entry in TRPC4(-/-) mice interferes with increase in lung microvascular permeability. Circ Res 91:70-76.
Mouse lung endothelial cells were labelled with a rat anti-mouse PECAM-1 (CD31) antibody (BD Pharmingen), and endothelial cells isolated using the Dynabeads® M-450 sheep anti-rat IgG. Isolated cells were washed, detached from the beads using trypsin treatment for 3 minutes, and put in culture.

Yeh JR et al. (2000) The antiangiogenic agent TNP-470 requires p53 and p21CIP/WAF for endothelial cell growth arrest. Proc Natl Acad Sci 97(23):12782-12787.
Dynabeads® M-450 sheep anti-rat IgG and rat anti-mouse E-selectin (CD62E, BD Pharmingen) were used for isolation of mouse pulmonary (lung) endothelial cells.

Answer Id: E6143

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47cc5a31edb3b1900cab4d583113cb66_FAQ

Application Note: Transcript profiling using the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit

Product Literature

0e4f5cc9f4f3f7f1651a6b9f9214e5b1_Brochures

Manual: ABI 433A Peptide Synthesizer: User Guide Volume 2 of 2 – Appendices: Rev D (English )

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Version: 20 Jun 2007
5a5766dd9760bfbfb73bfb50a66ae53d_Manuals

User Guide: GlobalFiler Express PCR Amplification Kit (English)

Manual / Product Insert

Version: 4477672 Rev. C (09May2014)
fad81a058424bc838f04871017bd142b_Manuals

User Guide: GlobalFiler PCR Amplification Kit - for use with 200 reaction kit & 1000 reaction kit

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Version: 4477604 Rev. C (04Sep2014)
022a50d5a53f9f27b285e58d37441bdd_Manuals

Activity Assay Kinase Selectivity Data

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fd5c905bcd8c3348ad1b35d7231ee2b1_Brochures

Additional Document: VariantSEQr™ Resequencing Primers: List of Available Genes: Miscellaneous

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f48c04ffab49ff0e5d1176244fdfb65c_Brochures

User Guide: Yfiler Plus PCR Amplification Kit

Manual / Product Insert

Version: 4485610 Rev.B (30Oct2014)
f501d2693c06f905f4c210b495748a79_Manuals

Manual: FastMoc™ Chemistry: HBTU Activation in Peptide Synthesis on the Model 431A: Model 431A Peptide Synthesizer (English )

Manual / Product Insert

Version: 20 Jun 2007
14ddc5c5bbb2f48452a62d6b3830672b_Manuals

User Guide: AmpFLSTR NGM SElect Express PCR Amplification Kit - for use with 200 Reaction Kit

Manual / Product Insert

Version: 4474109 Rev C (20Jun2014)
3ab2501f57ad3a624edf8599dc6237d5_Manuals

Manual: FastMoc™ 1.0 on the Model 431A: Model 431A Peptide Synthesizer: User Bulletin No. 36 (English )

Manual / Product Insert

Version: 20 Jun 2007
b678b443a0d40136143d792a9325d8af_Manuals