What is the purpose of immunoblotting (Western blotting)?
Explanation: The correct answer is C) Detecting and analyzing specific proteins. Immunoblotting, also known as Western blotting, is commonly used to detect and analyze specific proteins in a complex sample. It involves the separation of proteins by electrophoresis, followed by their transfer onto a membrane. The membrane is then probed with specific antibodies to visualize and analyze the target protein(s) of interest.
Which technique is typically used for protein separation prior to immunoblotting?
Explanation: The correct answer is C) Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Prior to immunoblotting, proteins are typically separated by SDS-PAGE. SDS-PAGE is a technique that separates proteins based on their molecular weight. The proteins are denatured, treated with SDS to give them a negative charge, and then subjected to electrophoresis through a polyacrylamide gel.
Which step of immunoblotting involves the transfer of proteins from the gel to a membrane?
Explanation: The correct answer is D) Protein transfer. After protein separation by SDS-PAGE, the proteins need to be transferred from the gel onto a membrane. This process is called protein transfer or blotting. It is typically done using electroblotting or semi-dry blotting methods, where an electric current is applied to facilitate the transfer of proteins from the gel to the membrane.
What is the purpose of blocking in immunoblotting?
Explanation: The correct answer is A) Preventing nonspecific binding of antibodies. Blocking is a crucial step in immunoblotting. It involves incubating the membrane with a blocking agent, such as bovine serum albumin (BSA) or nonfat milk, to prevent nonspecific binding of antibodies to the membrane. Blocking helps reduce background signal and ensures that the antibodies used for detection specifically bind to the target protein(s) of interest.
What is the detection method commonly used in immunoblotting?
Explanation: The correct answer is C) Chemiluminescence. Chemiluminescence is commonly used as a detection method in immunoblotting. It involves the use of enzyme-conjugated secondary antibodies that generate light upon reaction with a chemiluminescent substrate. The emitted light is detected using specialized imaging systems or X-ray film, allowing the visualization and quantification of the target protein bands on the membrane.