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Marker Gene Monthly Newsletter

April, 2005

Volume 5, Number 4

© Copyright MGT, Inc., 2007. Published by Marker Gene Technologies, Inc., The University of Oregon Riverfront Research Park, 1850 Millrace Drive, Eugene, Oregon 97403-1992 USA. All rights reserved. For information on the use or copying of the material contained in this document, please contact us at techservice@markergene.com. Please see below for subscription information and updates. This newsletter is labeled as an ADVERTISEMENT in accordance with the CAN-SPAM act of 2003, S.877 Public Law: 108-187.

Measuring Gene Regulation Using Marker Genes.

Transcription factors stimulate or repress gene expression of genes by interacting with promoter sequences. Their influence on the rate of protein production from downstream genes iscentral to the function of the cell. Until recently, it has been difficult to accurately monitor the effect of transcription factor concentration and eventual gene expression inside living cells. Recently, several studies have utilized expression of marker genes (like green, cyan, yellow or red fluorescent proteins (GFP, CFP, YPF, RFP) linked to either transcription factors (inducers or repressors) or to activatable promoters, to monitor the levels of both the transcription factor and the expressed protein, simultaneously inside individual cells. Theoretically the levels of gene expression should be proportional to the concentration of transcription factors that are activated. But most genes exist inside “gene networks” and are influenced by both intrinsic (gene expression machinery) and extrinsic (environment, cell cycle) factors. The resulting “noise” found in gene expression levels is a matter of importance to ultimately understanding the complex systems involved in cell growth and differentiation in living organisms and optimizing protein production. Work from the laboratory of Michael Elowitz and collaborators at CalTech, The Weizmann Institute (Israel), and McGill University (Canada) used YFP linked to a repressor and CFP linked to the target promoter to monitor gene transcription and correlate these to the levels of repressor inside living bacterial cells. Work from Alexander van Oudenaarden’s laboratory at the Department of Biophysics at MIT used a more complex network of CFP, YFP and RFP linked to the lacI operon with IPTG induction for CFP, Tet repressor for YFP and lambda repressor promoter controlling RFP (constitutive) in E. coli to monitor the levels of three genes simultaneously. In these cases, and others, it was found that extrinsic factors had a slightly higher influence than intrinsic factors on downstream gene expression and the network connectivity of gene networks (“noise”) had a greater effect than instrinsic expression of the gene itself. For the first time, marker genes are allowing researchers to accurately monitor such complex systems in vivo. For more information about these techniques, see ther references below, or visit our website.

N. Rosenfeld, J. W. Young, U. Alon, P. S. Swain, M.B. Elowitz (2005) “Gene Regulation at the Single-Cell Level” Science 307(5717): 1962-1965.

M. B.Elowitz, A. J.Levine, E. D.Siggia. P. S.Swain (2002) “Stochastic Gene Expression in a Single Cell” Science 297(16): 1183-1186.

F.J. Isaacs, W. J. Blake, J. J. Collins (2005) “Signal Processing in Single Cells” Science 307 (5717): 1886-1888.

J. M. Pedraza, A.van Oudenaarden (2005) “Noise Propagation in Gene Networks” Science 307(5717): 1965-1969.

Leucine Aminopeptidase as a Measure of Hepatic Function.

Leucine aminopeptidase (L-Leucyl-peptide hydrolase, [3.4.11.1], LAP) is a proteolytic exopeptidase that hydrolyzes the peptide bond adjacent to a free amino group next to L-leucine. It is called leucine aminopeptidase because it rapidly catalyzes the hydrolysis of leucine containing peptides and proteins at specific sites next to leucine amino acids. Determination of microsomal leucine amino peptidase activity in serum is of clinical significance to diagnose liver (hepatic) dysfunction, since serum LAP levels are elevated in obstructive jaundice, liver cirrhosis, liver carcinoma, systemic lupus erythematosus (SLE) and also during the later part of pregnancy. LAP is normally found inside liver cells (hepatocytes) but is released into the blood after damage to liver cells from drugs or infection (i.e., hepatitis). Measurement of its level in blood serves as an indicator of liver damage or the presence of tumors that arise in the liver possibly providing a tumor marker. Unlike other liver enzymes, LAP can also be measured in the urine, providing a more convenient diagnostic protocol. Leucine amino-peptidase is also extensively used in determination of the amino acid sequence of proteins and peptides, as well as for the determination of aminopeptidase activity in bacteria and marine organisms.

Marker Gene now provides the ultra-sensitive substrate L-Leucine 7-amido-4-methylcoumarin hydrochloride; Leu-AMC.HCl (M1060) (also called Aminopeptidase Substrate I) for use in fluorometric detection of this enzyme activity. The enzyme releases a bright blue fluorescent dye, 7-amino-4-methylcoumarin (AMC, M1059) upon enzyme activity (excitation 380nm emission 460nm). Sensitivity of this enzyme using fluorescence detection is greatly increased over other methods such as 4-Methoxy-naphthylamide (pNA) assays. In addition, AMC by the Ames Test has been shown to be a non-mutagenic chemical. For more information about these important peptidase assays visit our website or see the references below.

Smith GP; MacGregor RR; Peters TJ. (1983) “Subcellular localisation of leucine aminopeptidase in human polymorphonuclear leukocytes.” Biochim Biophys Acta 728(2): 222-7.

L. Riemann,M. Søndergaard (2004) “Profiles of bacterial community composition and metabolic potential through a plug-flow bioreactor fed with lake water” Journal of Plankton Research 26(8): 973-978.

Stepaniak L. (2000) “Comparison of different peptidase substrates for evaluation of microbial quality of aerobically stored meats.” J Food Prot. 63(10): 1447-9.

Bergmeyer, H.U., Methods of Enzymatic Analysis Vol. I, 26, 1975, Academic Press, New York.

Irvine GB ; Ennis M ; Williams CH (1990) “Visual detection of peptidase activity using fluorogenic substrates in a microtiter plate assay.” Anal Biochem 185(2): 304-307.

Cell Surface Engineering for Molecular Recognition.

Cells display on their surfaces a complex mixture of receptors and ligands that mediate cell adhesion, immune recognition and cell communication. Viruses also find convenient attachment points on these cell surface receptors. These ligands and receptors are often decorated with sugar polymers (oligosaccharides, glycans) whose structures are quite specific for attachment or communication. Recent work by Dr. Carolyn Bertozzi and co-workers at the University of California-Berkeley have centered on engineering new, and interesting receptors on cell surfaces by feeding the cells a sugar analog (Man-Lev) that they incorporate into their cell surface oligosaccharides as a modified version of the sugar sialic acid (5-NAcNeu). Since this sugar is usually found on the very outermost edges of the cell surface glycoproteins, the modified sugar that contains an aldehyde function can then be used to attach a second molecule, like biotin-hydrazide (M0128), thereby making a new “synthetic” cell surface receptor. Typically cells in culture are simply incubated with 25 mM N-levulinoyl-D-mannosamine (ManLev) or even more effectively with 25 µM ManLev tetraacetate. These ketone-containing monosaccharides serve as substrates in an oligosaccharide synthesis pathway, resulting in ketone-tagged cell-surface oligosaccharides. Other sugars, like GlcLev, have not been found to function as effectively for such modifications. For more information about these very interesting, new reagents and methods for cell surface labeling, please see the references below or visit our website.

Karema K.J., Bertozzi C.R., (1998) "Chemical Approaches to Glycobiology and Emerging Carbohydrate-Based Therapeutic Agents." Curr. Opin. Chem. Biol. 2: 49.

Yarema K.J., Mahal L.K., Bruehl R.E., Rodriguez E.C., Bertozzi C.R., (1998) "Metabolic delivery of ketone groups to sialic acid residues. Application To cell surface glycoform engineering." J. Biol. Chem. 273: 31168-31179.

Mahal L.K., Yarema K.J., Bertozzi C.R., (1997) "Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis." Science 276: 1125-1128.

Jacobs C.L., Yarema K.J., Mahal L.K., Nauman D.A., Charters N.W., Bertozzi C.R., (2000) "Metabolic labeling of glycoproteins with chemical tags through unnatural sialic acid biosynthesis." Methods Enzymol. 327: 260-275.

Charter N.W., Mahal L.K., Koshland D.E., Jr, Bertozzi C.R., (2000) "Biosynthetic incorporation of unnatural sialic acids into polysialic acid on neural cells." Glycobiology 10: 1049-1056 (2000).

Sampson N.S., Mrksich M., Bertozzi C.R. (2001) "Surface molecular recognition." Proc Natl Acad Sci U S A 98, 12870-1.

Yarema K.J., (2001) “Directions in carbohydrate engineering: a metabolic substrate-based approach to modify the cell surface display of sialic acids." Biotechniques 31: 384-393.

Antifade Reagents for Fluorescence Microscopy and Analysis.
Fading/bleaching of labeled specimens is a major problem in fluorescence microscopy. The high power and focused beam of a laser-scanning confocal microscope (LSCM) can cause rapid fading of a specimen’s fluorescence as compared to a conventional epifluorescent microscope which essentially bathes the entire specimen in lower-power, wide-beam excitatory light. Fluorophores are essentially destroyed by the high energy light during the observation process. The use of antifade reagents can significantly slow this fading process, allowing longer observation times in fluorometry and improved pattern recognition. Many factors influence the fluorescence intensity and bleaching of fluorophores, including the intensity (power) of excitatory light, the pH of the base solution or the embedding medium and the presence of other substances that quench fluorescence. Several theories have been proposed for the cause of fading/bleaching, including the damaging effects of oxygen, free-radicals, or protein denaturation. The most commonly studied fluorophore for fading/bleaching is FITC (fluorescein isothiocyanate). A few commonly used (and most effective) antifade reagents and their advantages/disadvantages are listed below. p-Phenylenediamine (PPD) is one of the most effective antifade reagents, although it suffers from photo/thermosensitivity, and toxicity that make it unsuitable for in vivo studies. Krenik et al., 1989 suggests the optimal PPD antifade mixture is a solution of 90% glycerol:10% PBS with PPD concentrations between 2mM and 7mM and a final pH of 8.5 to 9.0. A p-Phenylenediamindihydrochloride-Solution (10 ml PBS containing 100mg PPD, pH 8.0 adjusted with Carbonate-Bicarbonate-Buffer to pH 9.6, mixed with 90ml Glycerol and sterile filtered, stored dark at –20^oC) has also been used with success. N-propyl gallate (NPG) is non-toxic, and photo/thermo-stable but not as effective as PPD (Krenik et al., 1989) so that it can be used for in vivo studies. Recommended concentrations seem to fall in the range of 3mM to 9mM and a glycerol base also appears to be useful. If included in a mounting medium, a 5% n-propyl-gallate in a 1:1 glycerol : phosphate buffer saline mixture has been shown to work well with FITC and TRITC fluorochromes. The anti-oxidant takes time to dissolve at room temperature, so it is best to make it up the night before use. 1,4-diazobicyclo[2,2,2]-octane (DABCO) is a stable, non-ionizing, cheap and readily available antifade reagent for use in in vivo studies. Finally, ascorbic acid (Vitamin C) has also been used as an antifade reagent in cultured cells with success. For more information about these reagents for preventing fading of your fluorescently labeled samples, see the references below or visit our website.

Bock G., Hilchenbach M., Schauenstein K., Wick, G. “Photometric analysis of antifading reagents with laser and conventional illumination sources.” J. Histochem. & Cytochem. 33(7): 699-705, 1985.

Cytometry 19:177-182 (1995): "Analysis of antifading reagents for fluorescence microscopy."

Johnson G.D. and De C Nogueira Araujo G.M. A simple method for reducing the fading of immunofluorescence during microscopy. J. Immunol. Methods. 43:349, 1981.

Johnson G.D., Davidson R.S., McNamee K.C., Russell G., Goodwin D., and Holborow E.J. Fading of Immunofluorescence during microscopy: a study of the phenomenon and its remedy. J. Immunol. Methods. 55:231, 1982.

Krenik K.D., Kephart G.M., Offord K.P., Dunnette S.L. and Gleich, G.J. Comparison of antifading reagents used in immunofluorescence. J. Immunol. Methods. 117:91-7, 1989.

Longin A., Souchier C., French M., Bryon PA. Comparison of anti-fading agents used in fluorescence microscopy: image analysis and laser confocal microscopy study. J. Histochem. & Cytochem. 41(12):1833-40, 1993 Dec.

Compare Our Quality.
Marker Gene strives to offer our customers products of the highest quality and at the best possible prices. Our years of experience allow us to provide timely products for less cost to you. See our latest Price Comparison Chart that compares our prices with those from several alternate sources, to see if you can save money by switching to Marker Gene (http://www.markergene.com/crossref.htm). Or visit our website at www.markergene.com and click on the link “COMPARE”. We think you will appreciate our efforts to keep costs low and maintain excellent quality of our products for your research. For more information about any of our products, simply telephone us toll free at 1-888-218-4062 or contact us by e-mail at techservice@markergene.com. We will be happy to send you more about our products and their specifications.

CONTRACT RESEARCH@markergene.com

Marker Gene Technologies, Inc. has the expertise to perform contract research with you on your project. We have worked with many biotechnology and pharmaceutical companies on successful, proprietary and patented projects.

Contract Research and Development Capabilities in the following areas:

Established in 1993 at the University of Oregon Riverfront Research Park.

Screening Assay Development for HTS and uHTS

Chemical and Cellular Assays – High-Content Screening.

DNA/RNA (genomics) and protein (proteomics) labeling and assay development.

Pharmaceutical Intermediates - design, synthesis, and in vitro testing in mammalian cell culture.

Specializing in Carbohydrate, Lipid, Peptide, and Nucleic Acid Chemistries.

Fully equipped laboratories (Biochemistry, Chemical Synthesis, Tissue Culture, Analytical).

Confidentiality, help in patent preparation and filings.

Contact us by telephone at (888) 218-4062 or (541) 342-3760 or FAX us at (541) 342-1960 or you can write to us at Contract Research, Marker Gene Technologies, Inc., 1850 Millrace Drive, Eugene, Oregon 97403-1992 or contact us by e-mail at: techservice@markergene.com

Marker Gene Accepts Major Credit Cards.

Place your orders now, using Master Card or Visa and save time and money! Our Customer Assistance Staff can now accept either Master Card or Visa Credit Card orders, securely by telephone (toll-free) at 1-888-218-4062 (Domestic orders only). We will continue to accept Institutional Purchase Orders for our products, online or by FAX at 1-541-342-1960. International customers should contact us by e-mail, post or telephone for more information about International Distributors and ordering. For information on pricing for individual products, or for a quote on bulk quantities of our products or kits, please contact our technical assistance staff at techservice@markergene.com. We will be happy to assist you.

To add your name to our WebNewsletter mailing list, please use the following link: subscribe@markergene.com.

To unsubscribe from our mailing list, please use the following link: unsubscribe@markergene.com. Your e-mail address will be deleted in 2-3 business days.


	If you are having trouble viewing this email, click on this link. To order any of our products, please go to our ORDER FORM or visit one of our distributors:
	Marker Gene Monthly Newsletter April, 2005 Volume 5, Number 4 © Copyright MGT, Inc., 2007. Published by Marker Gene Technologies, Inc., The University of Oregon Riverfront Research Park, 1850 Millrace Drive, Eugene, Oregon 97403-1992 USA. All rights reserved. For information on the use or copying of the material contained in this document, please contact us at techservice@markergene.com. Please see below for subscription information and updates. This newsletter is labeled as an ADVERTISEMENT in accordance with the CAN-SPAM act of 2003, S.877 Public Law: 108-187.
	Measuring Gene Regulation Using Marker Genes. Transcription factors stimulate or repress gene expression of genes by interacting with promoter sequences. Their influence on the rate of protein production from downstream genes iscentral to the function of the cell. Until recently, it has been difficult to accurately monitor the effect of transcription factor concentration and eventual gene expression inside living cells. Recently, several studies have utilized expression of marker genes (like green, cyan, yellow or red fluorescent proteins (GFP, CFP, YPF, RFP) linked to either transcription factors (inducers or repressors) or to activatable promoters, to monitor the levels of both the transcription factor and the expressed protein, simultaneously inside individual cells. Theoretically the levels of gene expression should be proportional to the concentration of transcription factors that are activated. But most genes exist inside “gene networks” and are influenced by both intrinsic (gene expression machinery) and extrinsic (environment, cell cycle) factors. The resulting “noise” found in gene expression levels is a matter of importance to ultimately understanding the complex systems involved in cell growth and differentiation in living organisms and optimizing protein production. Work from the laboratory of Michael Elowitz and collaborators at CalTech, The Weizmann Institute (Israel), and McGill University (Canada) used YFP linked to a repressor and CFP linked to the target promoter to monitor gene transcription and correlate these to the levels of repressor inside living bacterial cells. Work from Alexander van Oudenaarden’s laboratory at the Department of Biophysics at MIT used a more complex network of CFP, YFP and RFP linked to the lacI operon with IPTG induction for CFP, Tet repressor for YFP and lambda repressor promoter controlling RFP (constitutive) in E. coli to monitor the levels of three genes simultaneously. In these cases, and others, it was found that extrinsic factors had a slightly higher influence than intrinsic factors on downstream gene expression and the network connectivity of gene networks (“noise”) had a greater effect than instrinsic expression of the gene itself. For the first time, marker genes are allowing researchers to accurately monitor such complex systems in vivo. For more information about these techniques, see ther references below, or visit our website. N. Rosenfeld, J. W. Young, U. Alon, P. S. Swain, M.B. Elowitz (2005) “Gene Regulation at the Single-Cell Level” Science 307(5717): 1962-1965. M. B.Elowitz, A. J.Levine, E. D.Siggia. P. S.Swain (2002) “Stochastic Gene Expression in a Single Cell” Science 297(16): 1183-1186. F.J. Isaacs, W. J. Blake, J. J. Collins (2005) “Signal Processing in Single Cells” Science 307 (5717): 1886-1888. J. M. Pedraza, A.van Oudenaarden (2005) “Noise Propagation in Gene Networks” Science 307(5717): 1965-1969.
	Leucine Aminopeptidase as a Measure of Hepatic Function. Leucine aminopeptidase (L-Leucyl-peptide hydrolase, [3.4.11.1], LAP) is a proteolytic exopeptidase that hydrolyzes the peptide bond adjacent to a free amino group next to L-leucine. It is called leucine aminopeptidase because it rapidly catalyzes the hydrolysis of leucine containing peptides and proteins at specific sites next to leucine amino acids. Determination of microsomal leucine amino peptidase activity in serum is of clinical significance to diagnose liver (hepatic) dysfunction, since serum LAP levels are elevated in obstructive jaundice, liver cirrhosis, liver carcinoma, systemic lupus erythematosus (SLE) and also during the later part of pregnancy. LAP is normally found inside liver cells (hepatocytes) but is released into the blood after damage to liver cells from drugs or infection (i.e., hepatitis). Measurement of its level in blood serves as an indicator of liver damage or the presence of tumors that arise in the liver possibly providing a tumor marker. Unlike other liver enzymes, LAP can also be measured in the urine, providing a more convenient diagnostic protocol. Leucine amino-peptidase is also extensively used in determination of the amino acid sequence of proteins and peptides, as well as for the determination of aminopeptidase activity in bacteria and marine organisms. Marker Gene now provides the ultra-sensitive substrate L-Leucine 7-amido-4-methylcoumarin hydrochloride; Leu-AMC.HCl (M1060) (also called Aminopeptidase Substrate I) for use in fluorometric detection of this enzyme activity. The enzyme releases a bright blue fluorescent dye, 7-amino-4-methylcoumarin (AMC, M1059) upon enzyme activity (excitation 380nm emission 460nm). Sensitivity of this enzyme using fluorescence detection is greatly increased over other methods such as 4-Methoxy-naphthylamide (pNA) assays. In addition, AMC by the Ames Test has been shown to be a non-mutagenic chemical. For more information about these important peptidase assays visit our website or see the references below. Smith GP; MacGregor RR; Peters TJ. (1983) “Subcellular localisation of leucine aminopeptidase in human polymorphonuclear leukocytes.” Biochim Biophys Acta 728(2): 222-7. L. Riemann,M. Søndergaard (2004) “Profiles of bacterial community composition and metabolic potential through a plug-flow bioreactor fed with lake water” Journal of Plankton Research 26(8): 973-978. Stepaniak L. (2000) “Comparison of different peptidase substrates for evaluation of microbial quality of aerobically stored meats.” J Food Prot. 63(10): 1447-9. Bergmeyer, H.U., Methods of Enzymatic Analysis Vol. I, 26, 1975, Academic Press, New York. Irvine GB ; Ennis M ; Williams CH (1990) “Visual detection of peptidase activity using fluorogenic substrates in a microtiter plate assay.” Anal Biochem 185(2): 304-307.
	Cell Surface Engineering for Molecular Recognition. Cells display on their surfaces a complex mixture of receptors and ligands that mediate cell adhesion, immune recognition and cell communication. Viruses also find convenient attachment points on these cell surface receptors. These ligands and receptors are often decorated with sugar polymers (oligosaccharides, glycans) whose structures are quite specific for attachment or communication. Recent work by Dr. Carolyn Bertozzi and co-workers at the University of California-Berkeley have centered on engineering new, and interesting receptors on cell surfaces by feeding the cells a sugar analog (Man-Lev) that they incorporate into their cell surface oligosaccharides as a modified version of the sugar sialic acid (5-NAcNeu). Since this sugar is usually found on the very outermost edges of the cell surface glycoproteins, the modified sugar that contains an aldehyde function can then be used to attach a second molecule, like biotin-hydrazide (M0128), thereby making a new “synthetic” cell surface receptor. Typically cells in culture are simply incubated with 25 mM N-levulinoyl-D-mannosamine (ManLev) or even more effectively with 25 µM ManLev tetraacetate. These ketone-containing monosaccharides serve as substrates in an oligosaccharide synthesis pathway, resulting in ketone-tagged cell-surface oligosaccharides. Other sugars, like GlcLev, have not been found to function as effectively for such modifications. For more information about these very interesting, new reagents and methods for cell surface labeling, please see the references below or visit our website. Karema K.J., Bertozzi C.R., (1998) "Chemical Approaches to Glycobiology and Emerging Carbohydrate-Based Therapeutic Agents." Curr. Opin. Chem. Biol. 2: 49. Yarema K.J., Mahal L.K., Bruehl R.E., Rodriguez E.C., Bertozzi C.R., (1998) "Metabolic delivery of ketone groups to sialic acid residues. Application To cell surface glycoform engineering." J. Biol. Chem. 273: 31168-31179. Mahal L.K., Yarema K.J., Bertozzi C.R., (1997) "Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis." Science 276: 1125-1128. Jacobs C.L., Yarema K.J., Mahal L.K., Nauman D.A., Charters N.W., Bertozzi C.R., (2000) "Metabolic labeling of glycoproteins with chemical tags through unnatural sialic acid biosynthesis." Methods Enzymol. 327: 260-275. Charter N.W., Mahal L.K., Koshland D.E., Jr, Bertozzi C.R., (2000) "Biosynthetic incorporation of unnatural sialic acids into polysialic acid on neural cells." Glycobiology 10: 1049-1056 (2000). Sampson N.S., Mrksich M., Bertozzi C.R. (2001) "Surface molecular recognition." Proc Natl Acad Sci U S A 98, 12870-1. Yarema K.J., (2001) “Directions in carbohydrate engineering: a metabolic substrate-based approach to modify the cell surface display of sialic acids." Biotechniques 31: 384-393.
	Antifade Reagents for Fluorescence Microscopy and Analysis. Fading/bleaching of labeled specimens is a major problem in fluorescence microscopy. The high power and focused beam of a laser-scanning confocal microscope (LSCM) can cause rapid fading of a specimen’s fluorescence as compared to a conventional epifluorescent microscope which essentially bathes the entire specimen in lower-power, wide-beam excitatory light. Fluorophores are essentially destroyed by the high energy light during the observation process. The use of antifade reagents can significantly slow this fading process, allowing longer observation times in fluorometry and improved pattern recognition. Many factors influence the fluorescence intensity and bleaching of fluorophores, including the intensity (power) of excitatory light, the pH of the base solution or the embedding medium and the presence of other substances that quench fluorescence. Several theories have been proposed for the cause of fading/bleaching, including the damaging effects of oxygen, free-radicals, or protein denaturation. The most commonly studied fluorophore for fading/bleaching is FITC (fluorescein isothiocyanate). A few commonly used (and most effective) antifade reagents and their advantages/disadvantages are listed below. p-Phenylenediamine (PPD) is one of the most effective antifade reagents, although it suffers from photo/thermosensitivity, and toxicity that make it unsuitable for in vivo studies. Krenik et al., 1989 suggests the optimal PPD antifade mixture is a solution of 90% glycerol:10% PBS with PPD concentrations between 2mM and 7mM and a final pH of 8.5 to 9.0. A p-Phenylenediamindihydrochloride-Solution (10 ml PBS containing 100mg PPD, pH 8.0 adjusted with Carbonate-Bicarbonate-Buffer to pH 9.6, mixed with 90ml Glycerol and sterile filtered, stored dark at –20^oC) has also been used with success. N-propyl gallate (NPG) is non-toxic, and photo/thermo-stable but not as effective as PPD (Krenik et al., 1989) so that it can be used for in vivo studies. Recommended concentrations seem to fall in the range of 3mM to 9mM and a glycerol base also appears to be useful. If included in a mounting medium, a 5% n-propyl-gallate in a 1:1 glycerol : phosphate buffer saline mixture has been shown to work well with FITC and TRITC fluorochromes. The anti-oxidant takes time to dissolve at room temperature, so it is best to make it up the night before use. 1,4-diazobicyclo[2,2,2]-octane (DABCO) is a stable, non-ionizing, cheap and readily available antifade reagent for use in in vivo studies. Finally, ascorbic acid (Vitamin C) has also been used as an antifade reagent in cultured cells with success. For more information about these reagents for preventing fading of your fluorescently labeled samples, see the references below or visit our website. Bock G., Hilchenbach M., Schauenstein K., Wick, G. “Photometric analysis of antifading reagents with laser and conventional illumination sources.” J. Histochem. & Cytochem. 33(7): 699-705, 1985. Cytometry 19:177-182 (1995): "Analysis of antifading reagents for fluorescence microscopy." Johnson G.D. and De C Nogueira Araujo G.M. A simple method for reducing the fading of immunofluorescence during microscopy. J. Immunol. Methods. 43:349, 1981. Johnson G.D., Davidson R.S., McNamee K.C., Russell G., Goodwin D., and Holborow E.J. Fading of Immunofluorescence during microscopy: a study of the phenomenon and its remedy. J. Immunol. Methods. 55:231, 1982. Krenik K.D., Kephart G.M., Offord K.P., Dunnette S.L. and Gleich, G.J. Comparison of antifading reagents used in immunofluorescence. J. Immunol. Methods. 117:91-7, 1989. Longin A., Souchier C., French M., Bryon PA. Comparison of anti-fading agents used in fluorescence microscopy: image analysis and laser confocal microscopy study. J. Histochem. & Cytochem. 41(12):1833-40, 1993 Dec.
	Compare Our Quality. Marker Gene strives to offer our customers products of the highest quality and at the best possible prices. Our years of experience allow us to provide timely products for less cost to you. See our latest Price Comparison Chart that compares our prices with those from several alternate sources, to see if you can save money by switching to Marker Gene (http://www.markergene.com/crossref.htm). Or visit our website at www.markergene.com and click on the link “COMPARE”. We think you will appreciate our efforts to keep costs low and maintain excellent quality of our products for your research. For more information about any of our products, simply telephone us toll free at 1-888-218-4062 or contact us by e-mail at techservice@markergene.com. We will be happy to send you more about our products and their specifications.
	CONTRACT RESEARCH@markergene.com Marker Gene Technologies, Inc. has the expertise to perform contract research with you on your project. We have worked with many biotechnology and pharmaceutical companies on successful, proprietary and patented projects. Contract Research and Development Capabilities in the following areas: Established in 1993 at the University of Oregon Riverfront Research Park. Screening Assay Development for HTS and uHTS Chemical and Cellular Assays – High-Content Screening. DNA/RNA (genomics) and protein (proteomics) labeling and assay development. Pharmaceutical Intermediates - design, synthesis, and in vitro testing in mammalian cell culture. Specializing in Carbohydrate, Lipid, Peptide, and Nucleic Acid Chemistries. Fully equipped laboratories (Biochemistry, Chemical Synthesis, Tissue Culture, Analytical). Confidentiality, help in patent preparation and filings. Contact us by telephone at (888) 218-4062 or (541) 342-3760 or FAX us at (541) 342-1960 or you can write to us at Contract Research, Marker Gene Technologies, Inc., 1850 Millrace Drive, Eugene, Oregon 97403-1992 or contact us by e-mail at: techservice@markergene.com
	Marker Gene Accepts Major Credit Cards. Place your orders now, using Master Card or Visa and save time and money! Our Customer Assistance Staff can now accept either Master Card or Visa Credit Card orders, securely by telephone (toll-free) at 1-888-218-4062 (Domestic orders only). We will continue to accept Institutional Purchase Orders for our products, online or by FAX at 1-541-342-1960. International customers should contact us by e-mail, post or telephone for more information about International Distributors and ordering. For information on pricing for individual products, or for a quote on bulk quantities of our products or kits, please contact our technical assistance staff at techservice@markergene.com. We will be happy to assist you.
	To add your name to our WebNewsletter mailing list, please use the following link: subscribe@markergene.com. To unsubscribe from our mailing list, please use the following link: unsubscribe@markergene.com. Your e-mail address will be deleted in 2-3 business days.