in situ cell -free expression of unknown proteins
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
In situ cell-free expression of unknown proteins
Progres Meeting Madrid, 2012
1616 Genes on Chromosome 16
B-lymphocyte T-lymphocyte Leukocyte Epithelium Fibroblast
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Protein coding genes (n=862)
Entr
ies
More than 85 % of protein coding genes of chromosome 16 are expressed in lymphoid cells, epitelial cells and fibroblasts.
1616 Genes on Chromosome 16
B-lymphocyte T-lymphocyte Leukocyte Epithelium Fibroblast
Entries 538 244 263 491 329
50
150
250
350
450
550
Protein coding genes (n=862)
Entr
ies
More than 85 % of protein coding genes of chromosome 16 are expressed in lymphoid cells, epitelial cells and fibroblasts.
Mix two plasmids and enzymeOnly the Desired Product Survives
Master
Moving Genes by Recombination
Gateway Technology for cloning & subcloning
•Master Library Many libraries•Any vector can be modified to accept genes
•Master Library Many libraries•Any vector can be modified to accept genes
Testing Proteins Many Different Ways
Testing Single Proteins in Many Different Ways
Where is the protein
located in the cell?
Produce lots of
protein for experiment
s.
Turn the protein on and off in mammalian
cells.
Test the protein’s function in
mammalian cells.
What other proteins does this protein
interact with?
Perform assays using protein made in
bacteria.
Marcador 1Kb LRRC29 MC1R
KCNG4
TMEM170
GSG1LIGSF6 CKLFSF2
WFDC1
Insert Verification by Xho1 & BamHI Digestion
Detection of in situ cell-free protein expression
WB Beads Suspension Arrays (BBAs)
tagged protein ontoanti-GST beads
Cell-free expression of GST-tagged DNA
MixDetection
Anti-GST Anti-mouse
Coupling of anti-GST antibody to beads
Capture of GST-
re-aliquot
Beads Suspension Arrays