Selected methods in molecular biology 3 · Selected methods in molecular biology 3 Assist. ......

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