Practical 2. SDS and Western Blotting

How does SDS separate proteins?

According to molecular weight

What is an electric field used for?

To transfer the protein to the membrane

What is the membrane like?

Porous

What is immunoblotting?

The use of antibodies to identify target proteins

What are primary antibodies specific to?

The protein of interest

If primary antibodies bind to the membrane what does this indicate?

Protein of interest is present

What do secondary antibodies bind to?

Enzyme then added to primary antibody

What is the enzyme used for?

To detect the protein of interest

When do conjugated antibodies come in to play?

If protein of interst is present and bound by enzyme

What do conjugated antibodies do?

Catalyse conversion of substrate to product.

What do conjugated antibodies produce?

Detectable chemiluminescent signal where protein is bound.

What do comonents of the blotting sandwich need to be soaked in?

Transfer buffer

What does transfer buffer do?

Prevents gel from shrinking and distorting bands

What does blocking sollution contain?

Proteins with no affinity for proteins in sample or antibodies used.

What do the proteins in blocking solution do?

Bind to areas of blot no occupied by sample protein so when antibodies are later added they are prevented from binding non-specifically to membrane

What are examples of blocking solutions?

Non-fat dried milk and BSA

What is added to wash buffer to support blocking action?

TBST

What do secondary antibodies have to be?

Anti-primary antibody