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In situ cell -free expression of unknown proteins

In situ cell -free expression of unknown proteins. Progres Meeting Madrid, 2012. 16. Genes on Chromosome 16. 16. More than 85 % of protein coding genes of chromosome 16 are expressed in lymphoid cells , epitelial cells and fibroblasts. 16. Genes on Chromosome 16. 16.

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In situ cell -free expression of unknown proteins

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  1. In situ cell-free expression of unknownproteins Progres Meeting Madrid, 2012

  2. 16 Genes onChromosome 16 16 More than85 % of proteincoding genes of chromosome 16 are expressed in lymphoidcells, epitelial cells and fibroblasts.

  3. 16 Genes onChromosome 16 16 More than85 % of proteincoding genes of chromosome 16 are expressed in lymphoidcells, epitelial cells and fibroblasts.

  4. Gateway Technologyforcloning & subcloning Moving Genes by Recombination Master Only the Desired Product Survives Mix two plasmids and enzyme

  5. Testing Proteins Many Different Ways • Master Library  Many libraries • Any vector can be modified to accept genes

  6. Testing Single Proteins in Many Different Ways Perform assays using protein made in bacteria. What other proteins does this protein interact with? Where is the protein located in the cell? Test the protein’s function in mammalian cells. Produce lots of protein for experiments. Turn the protein on and off in mammalian cells.

  7. InsertVerificationby Xho1 & BamHIDigestion TMEM170 Marcador 1Kb WFDC1 MC1R LRRC29 KCNG4 IGSF6 CKLFSF2 GSG1L

  8. Detection of in situ cell-free proteinexpression WB BeadsSuspensionArrays (BBAs)

  9. Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y BeadsSuspensionArrays Coupling of anti-GST antibody to beads Anti-GST Anti-mouse Mix Capture of GST- Detection re-aliquot tagged protein onto anti-GST beads Cell-free expression of GST-tagged DNA

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