Mobility shift assay and footpriting of DNA binding proteins

Aim

DNA-binding proteins are crucial players in the regulation of gene expression. In this course we will discuss different methods for studying DNA-binding proteins and the theoretical background to different classes of DNA-binding motifs, their properties and specific requirements.
In the practical part of the course we will focus on the performance of electrophoretic mobility shift assay (EMSA) and the DNA footprinting analysis, which are two techniques used for the determination of a DNA target motif and for analysis of the binding affinity. The participants will have the opportunity to bring their own extracts for analysis by EMSA.
The course will be experimentally intense and will include the following steps: Preparation of cell extracts and protein concentration determination. Labeling of DNA targets with radioactive isotopes. Binding reactions and analysis of protein binding by electrophoresis on polyacrylamide gels. Detection by phosphorimaging. Testing of binding specificity by competition analyses. Antibody supershift analysis. DNA footprint reactions and analysis on sequencing grade polyacrylamide gels.
We will address the concepts of binding specificity, binding affinity, stoichiometry and cooperativity both in practice and in theory, and we will discuss other methods for analyzing DNA-binding proteins: chromatin immunoprecipitation (ChIP), methylation protection, and methylation interference assays.

Location & Organization

Organizer

FLÄK - The Research School in Pharmaceutical Sciences

Course Director

Marita Cohn

Location / venue

Lund University (Faculty of Science)

Timing & Workload

Duration 1 week
ECTS points 3
Frequency Annual
 

Examination yes

Criteria

Is the course taught in English? yes
Is documentation available? (book, syllabus)? yes
Is the course open for external researchers? yes

More Information

http://www.biol.lu.se/cellorgbiol/postgrad/courses/course_14.html