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  • Biology-Online • View topic - Method for enhancing solubility of recombinant protein
    economy etc However when expressed in e coli most recombinant proteins deposit in the form of insoluble and inactive inclusion bodies In vitro renaturation is a complicated and low efficient process The purpose of the invention can be achieved by the following method which comprises introducing the leader sequence at 5 UTR untranslatable region of tobacco mosaic virus TMV genome into an E coli expression vector between the promoter region and the ribosome binding site rbs thereby forming a novel expression vector which increases the yield of soluble biologically active recombinant products and effectively avoids or improves the formation of inclusion bodies of recombinant proteins In the first aspect the invention provides an E coli expression vector comprising an omega leader sequence between the promoter region and ribosome binding site In a preferred embodiment the omega leader sequence comprises the sequence of SEQ ID NO 2 In a preferred embodiment said expression vector comprises a core regulatory region having the sequence of SEQ ID NO 1 In a preferred embodiment said expression vector is a GST fusion expression vector In the second aspect the invention provides an E coli comprising the E coli expression vector of the invention In the second aspect the invention provides a method for improving an E coli expression vector comprising the steps of 1 providing an E coli expression vector which comprises an promoter region and ribosome binding site 2 inserting an omega leader sequence between said promoter region and ribosome binding site thereby forming an expression vector which comprises said omega leader sequence In a preferred embodiment said method further comprises the step of 3 inserting the GST coding sequence the multiple cloning sites and the histidine tag coding region orderly into said vector downstream of the ribosome binding site thereby forming a GST

    Original URL path: http://www.biology-online.org/biology-forum/about43084.html (2016-02-17)
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  • Biology-Online • View topic - Describe the difference between these terms
    reply Reply with quote d9esco d9esco Posts 10 Joined Wed Sep 23 2015 8 15 pm Describe the difference between these terms Sun Nov 08 2015 8 05 pm StereoIsomer Monomer isomer and polymer Please also list other words using different prefixes to the word Isomer Post a reply Jump to Select a forum General Biology General Discussion Cell Biology Molecular Biology Zoology Discussion Evolution Microbiology Bioinformatics Human Biology Botany

    Original URL path: http://www.biology-online.org/biology-forum/about43077.html (2016-02-17)
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  • Biology-Online • View topic - most complex abiotic (nonliving) molecules
    crystals but these generally only have a few unique elements repeated over and over again Thanks Reply with quote EnricoPallazzo EnricoPallazzo Posts 16 Joined Sun Dec 04 2011 6 13 pm Re most complex abiotic nonliving molecules Sat Oct 31 2015 6 16 pm Lignin comes into my mind Lignins are formed via radical induced coupling of phenolic compounds and support the strength of plant cell walls However they are part of plant tissues in trees and they might not exactly meet your definition But the lignin in a single tree is believed to be one continuous growing structure Thus it can be seen as one large molecule Reply with quote mattw mattw Posts 41 Joined Wed Jan 31 2007 4 39 pm Re most complex abiotic nonliving molecules Sat Oct 31 2015 6 25 pm That s great Living nature wins again Lignin is a huge molecule Are there any complex molecules that develop without living syatems Reply with quote claudepa claudepa Posts 27 Joined Tue Nov 03 2015 12 43 pm Re most complex abiotic nonliving molecules Wed Nov 04 2015 5 10 pm HI Its seems to me very difficult to know on earth if an abiotic

    Original URL path: http://www.biology-online.org/biology-forum/about43051.html (2016-02-17)
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  • Biology-Online • View topic - Ni-NTA-Columns
    stringency of the binding and washing buffer and eluted the last time with 200mM Imidazol in the first step and with 350 mM Imidazol in the second elution step But there are still contaminants What is the maximum Imidazol concentration in the elution buffer or are there any other tricks to get rid of these contaminants Reply with quote blcr11 blcr11 Posts 672 Joined Fri Mar 30 2007 4 23 am Thu Feb 14 2008 12 09 pm If you re using an fplc instrument you re better off using a gradient of imidazole for elution Then your stuff will elute at whatever minimal concentration of imidazole that suits it You can also try using some other chelation matrix USB for one sells a silica based Ni resin they claim gives lower non specific binding compared to either IMAC or agarose type resins Reply with quote BioLad BioLad Posts 14 Joined Wed Nov 21 2007 6 58 pm Re Ni NTA Columns Thu Feb 14 2008 4 49 pm I m with Piefke here about gradients I used USB s PrepEase kits and they work great http www usbweb com category asp spec 5 id 78794 Reply with quote piefke piefke Posts 24 Joined Tue Sep 18 2007 8 41 am Re Ni NTA Columns Thu Feb 21 2008 8 09 am thank you I think I will try the other columns Reply with quote chance chance Posts 10 Joined Wed Sep 09 2015 3 39 am Website Re Ni NTA Columns Thu Oct 15 2015 9 13 am 1 Imidazole concentration is too high When eluting with imidazole each gradient must elute completely 2 The cleaning of sample is not sufficient unable to remove unbound contaminants You can extend the cleaning time and increase the imidazole concentration to improve

    Original URL path: http://www.biology-online.org/biology-forum/about13041.html (2016-02-17)
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  • Biology-Online • View topic - experiences with ni-affinity protein purification??
    values or to elute the protein with EDTA Has anybody any experiences with the elution of protein from nickel collumns in this ways I fear that my enzyme will denaturate when I decrease the pH Or is it possible that the pH has not to be decreased as much After elution with EDTA it must be possible to get rid of the EDTA with dialysis right Thanks for comments or ideas Reply with quote blcr11 blcr11 Posts 672 Joined Fri Mar 30 2007 4 23 am Sun Jun 29 2008 12 03 am Ni columns have been eluted by either low pH or with EDTA I have no direct experience with it but it has been known to work When you assayed your protein in the presence of imidazole you checked to make sure the pH was what you thought it was after adding imidazole I presume Imidazole especially 250 mM can change the pH of your buffer Imidazole can also carry in contaminants like zinc If you have a metalloenzyme inhibited by zinc that could contribute to loss of activity in the presence of imidazole which wouldn t necessarily be due to imidazole You can t dialyze away the imidazole and restore activity Reply with quote rdxfun rdxfun Posts 1 Joined Thu Jan 29 2009 12 19 am Re experiences with ni affinity protein purification Thu Jan 29 2009 12 34 am Your protein is inactive due to the high concentration of imidazole Try to use a cobalt based resin and that should do the trick You will extract and wash in absence of imidazole and then purify in low concentration s of imidazole Reply with quote chance chance Posts 10 Joined Wed Sep 09 2015 3 39 am Website Re experiences with ni affinity protein purification Thu Oct

    Original URL path: http://www.biology-online.org/biology-forum/about13903.html (2016-02-17)
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  • Biology-Online • View topic - Total protein concentration before Elisa
    I am going to do elisa which i get from here http www creative diagnostics com Con pment html but i don t know what is the point of measuring the total protein concentration before Elisa The most papers i have reviewed using Elisa in BALf have results in pg ml or ng ml or ug ml and they have not performed an assay for the total protein concentration Reply

    Original URL path: http://www.biology-online.org/biology-forum/about41911.html (2016-02-17)
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  • Biology-Online • View topic - Restriction Digesion
    for Restriction Endonuclease digesion A 37 C water bath B 37 C PCR program Reply with quote MOmarKhan MOmarKhan Posts 1 Joined Mon Apr 07 2014 2 05 am Mon Apr 07 2014 2 09 am Both of them are okay But i would go for Thermo cycler instead of water bath Reply with quote Helics Helics Posts 42 Joined Fri Oct 12 2012 9 10 am Tue Apr 08 2014 4 26 am Dear scientist Jack Bean I need your response Reply with quote JackBean JackBean Posts 5694 Joined Mon Sep 14 2009 7 12 pm Sun May 04 2014 12 04 pm thanks for considering me a scientist I don t see much difference if you re sure that the temperature iscorrectly set However with thermocycler or thermoblock you do not have to worry that you will spill your samples Reply with quote Javedz2001 Javedz2001 Posts 6 Joined Sun Oct 11 2015 4 32 pm Sun Oct 11 2015 5 19 pm Water bath is NOT recommended because after time the sample will evaporate and stick on the top of the cap This will change the buffer composition Always use PCR machine with heated lid or dry incubator

    Original URL path: http://www.biology-online.org/biology-forum/about38450.html (2016-02-17)
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  • Biology-Online • View topic - TAE Buffer preparation
    4 32 pm Sun Oct 11 2015 5 14 pm DO NOT add NaOH to any of the electrophoresis buffer Strong acid or base can not be use to adjust the pH of electrophoresis buffer It will have increase the current and heat up the runner buffer Always use Acetic acid boric acid phosphoric acids for adjusting the pH 50x TAE needs the following 242 grams of Tris base 57

    Original URL path: http://www.biology-online.org/biology-forum/about41588.html (2016-02-17)
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