Application Note: Determination of Different Classes of Pesticide Residues in Processed Fruits and Vegetables by LC-MS Using the TSQ Quantum Ultra According to EU Directive 91/414 EEC

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Product Insert: Anza 91 Acc65I

Manual / Product Insert

Version: Rev A (21 July 2015)
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Pierce™ SNAP-91 Antibody (AP180-I)

MSDS

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Pierce™ SUMO3 Antibody (76AT630.91.31)

MSDS

20f05c19257dc9a9b95a9279ffe9175a_MSDS

CD5 Antibody, RPE conjugate (25.91)

MSDS

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78a994dd901a192eb466d026efdd077d_MSDS

Application Note: Choline and Acetylcholine

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Application Note: Determination of Aluminum in Complex Matrices Using Chelation Ion Chromatography

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Application Note: Determination of Diethanolamine and Triethanolamine in Surface Finishing, Wastewater and Scrubber Solutions

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Application Note: Ion Chromatography of Lanthanide Metals

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Application Note: Determination of Chromium by Ion Chromatorgraphy

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

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Answer

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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Answer

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

I am using the LightShift Chemiluminescent EMSA kit. How do I make the non-specific bands go away?

Product FAQ

Answer

Non-specific bands may be reduced or eliminated by optimizing the binding reactions. Strong non-specific bands can often be mistaken for a positive result but will not be blocked by the addition of cold competitor probe. The following are examples of changes that MAY help reduce non-specific bands:

• Reduce the amount of protein extract in the binding reaction.
• Increase the amount of non-specific competitor DNA [i.e., poly(dI-dC)·poly(dI-dC)].
• Use a different non-specific competitor DNA [i.e., sonicated salmon sperm DNA or poly(dA-dT)·poly(dA-dT)].
• Preincubate the protein extract with non-specific competitor DNA before adding the biotinylated probe.
• Shorten or redesign the probe used in the experiment.

Answer Id: E8431

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be459a197a9a8cf29b4dc6ea782578cd_FAQ

A protein sample with many disulfide bonds, reduced with BME or DTT, is exhibiting smeary artifacts on a Tricine Gel. Are the samples insufficiently reduced?

Product FAQ

Answer

One potential explanation is that the protein sample is getting re-oxidized before the run is complete. 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 degrees C for 30 minutes, followed by 50 mM iodoacetic acid. Another method which inhibits oxidation is the addition of thioglycolic acid to the running buffer. The reference to this is described by Hunkapiller et al., Methods in 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 get self-oxidized over time and will promote sample re oxidation.

Answer Id: E10779

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