Difference between revisions of "Part:BBa K3795000:Experience"

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===Applications of BBa_K3795000===
 
===Applications of BBa_K3795000===
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In two immunoassays conducted one anti-dengue and one anti-Zika western blot with DME-C. The anti-dengue was to confirm that our  protein was really capable of detecting dengue virus, and the result was positive so we are really capable of detecting dengue. But to verify if cross reaction was occurring, we performed a anti-zika western blot, and a band appeared, although it can possibly indicate cross reaction, considering that the experiment was performed on a membrane used first to other western blot assay, there may have been an error related to washing, at the time the stripping was performed, the antibodies were not completely removed, and therefore when Zika's membrane was revealed, the result was not the expected. So, for a better precision, the experiment must be done again, with a new membrane, avoiding interference.  
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In order to evaluate if DME-C could recognize anti-Dengue antibodies, western blot analysis were performed with two different commercial antibodies: anti-Dengue NS1, and anti-Dengue envelope protein. Fortunately, both DME-C and DME-BR were capable of recognizing anti-Dengue E antibodies, showing that Ammit fullfiled at least its main function. However, the preliminary anti-Zika assays showed that the protein also recognized anti-Zika antibodies.
  
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https://2021.igem.org/wiki/images/e/e5/T--Rio_UFRJ_Brazil--rostoJuliano.png
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https://static.igem.org/mediawiki/parts/9/9f/T--Rio_UFRJ_Brazil--Anti-E-peq-denv.png
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Anti-Dengue Envelope (Invitrogen) protein Western blot analysis. We used  Anti - Mouse IRDye® 800CW (LI-COR), at 1:10,000, as the secondary antibody. M: Protein marker (Spectra™ Multicolor Low Range Protein, Thermo). 1 - 3: Different dengue virus lysate containing 30 μg of protein used as a positive control. 4: 17 μg of DME-C. 5: 17 μg of DME-BR.
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https://static.igem.org/mediawiki/parts/2/28/T--Rio_UFRJ_Brazil--Anti-Ns1-peq-Zika.png
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(1)e(2)MW Molecular Weight Marker, Unstained Protein Molecular Weight Marker; (3) 2.0μg DMEC sample; (4) 1.0μg DMEC sample; (5) Sample of 2.0μg DMEC; (6) DMEBR 2.0μg sample; (7) DMEBR 1.0μg sample; (8) DMEBR 0.5μg sample;
  
 
===User Reviews===
 
===User Reviews===

Revision as of 01:21, 22 October 2021


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Applications of BBa_K3795000

In order to evaluate if DME-C could recognize anti-Dengue antibodies, western blot analysis were performed with two different commercial antibodies: anti-Dengue NS1, and anti-Dengue envelope protein. Fortunately, both DME-C and DME-BR were capable of recognizing anti-Dengue E antibodies, showing that Ammit fullfiled at least its main function. However, the preliminary anti-Zika assays showed that the protein also recognized anti-Zika antibodies.

T--Rio_UFRJ_Brazil--Anti-E-peq-denv.png Anti-Dengue Envelope (Invitrogen) protein Western blot analysis. We used Anti - Mouse IRDye® 800CW (LI-COR), at 1:10,000, as the secondary antibody. M: Protein marker (Spectra™ Multicolor Low Range Protein, Thermo). 1 - 3: Different dengue virus lysate containing 30 μg of protein used as a positive control. 4: 17 μg of DME-C. 5: 17 μg of DME-BR.

T--Rio_UFRJ_Brazil--Anti-Ns1-peq-Zika.png (1)e(2)MW Molecular Weight Marker, Unstained Protein Molecular Weight Marker; (3) 2.0μg DMEC sample; (4) 1.0μg DMEC sample; (5) Sample of 2.0μg DMEC; (6) DMEBR 2.0μg sample; (7) DMEBR 1.0μg sample; (8) DMEBR 0.5μg sample;

User Reviews

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