Selected methods in molecular biology 3
Assist. Prof. Tomaž Bratkovič
University of Ljubljana, Faculty of Pharmacy
Gene expression at the protein level
• different levels of transcription
• codon usage (frequency)
• mRNA stability (higher level structures)
• post-transcriptional regulation (e.g. RNAi)
• posttranslational modifications
Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE)
Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE)
A discontinuous system (two-gel system:
stacking & resolving)
Western blot
Western blot
Western blot
Isoelectric focusing (IEF)
Example: recombinant EPO
Isoelectric focusing (IEF)
pH gradient gel preparation
2D electrophoresis
Differential 2D gel electrophoresis
Immunocytochemistry & immunohistochemistry
Direct vs. indirect detection
Enzyme-linked immunosorbent assay (ELISA)
Antibody (IgG) structure
Köhler, Milstein & Jerne, 1975
(Nobel prize in medicine and
physiology 1984)
Hybridoma technology
Hybridoma technology
Cell lysis and protein solubilization
Coimmunoprecipitation (pull-down assay)
Coimmunoprecipitation (pull-down assay)
1 maker 2 bead matrix NC 3 scrambled peptide as capture
molecule (NC) 4 peptide pull-down
1 2 3 4
potential binding partner
kDa
Ngubane NA et al. PLoS One 2013; 8(11): e77844.
separation
Size exclusion chromatography for analyses of protein complexes