3.6 Quantifying western blot data

What is the definition of ALS?

An adult-onset neurodegenerative disease characterized by progressive muscle weakness and loss of motorneurons in the brain and spinal cord.

What is the definition of western blot?

A widely used immunoblotting technique used to detect a specific protein in a sample of a tissue/cell type.

Based on what characteristic are proteins separated in a western blot?

Molecular weight

What is used to detect proteins of interest in western blot?

Probes

What is the first step of western blot?

Cell lysis

• Detergent disruption for culture cells

• Which fraction if a protein of interest?

What is step 2 in a western blot?

Protein separation

• Gel electrophoresis: movement of charges molecules in response to an electric field

• Polyacrylamide gel electrophoresis (PAGE): size selection of proteins

• SDS-PAGE: SDS helps denature proteins and adds an overall negative charge

What is step 3 in a western blot?

Protein transfer from gel onto membrane

What is step 4 in a western blot?

Protein detection

1. Blocking of unoccupied binding sites

2. Primary antibody against protein of interest

3. Secondary antibody against species of 1st AB (with flurophore)

4. Detection of signal

What is step 5 in a western blot?

Image analysis

• Signal intensity is directly proportional to concentration of the target protein

• Western blot normalization: housekeeping protein

What is the role of TDP-43?

TDP-43 regulates mRNA splicing, stability and translation as well as gene transcription. It’s normally localized in the nucleus, but sometimes it is localized in the plasma to carry out diverse cell functions.

What happens in ALS when there is a mutation of TDP-43?

It results in another splicing product. It is mislocalized from the nucleus to the cytoplasm and there are also insoluble aggregates present.

What are TDP-43 mutations in the C-terminal associated with?

Increased levels of insoluble protein aggregates and soluble TDP-43 contains no mutations.