Western Blotting, proteins and antibody methods Flashcards
(11 cards)
Primary and secondary antibodies
Primary - the one that binds to the epitope (part of antigen)
Secondary - the one that binds to the primary
Tertiary if another
Polyclonal antibodies
Polyclonal antibodies are a mixture of antibodies produced by different B cells that recognize and bind to multiple epitopes on the same antigen
Monoclonal antibodies
Monoclonal antibodies are laboratory-produced molecules designed to serve as substitute antibodies that can precisely target a specific antigen. They are made by identical immune cells that are all clones of a unique parent B cell, meaning they are all the same and recognize one specific epitope (part) of an antigen.
To produce monoclonal antibodies, scientists typically fuse a single B cell (that produces the desired antibody) with a myeloma (cancer) cell. This hybrid cell, called a hybridoma, can both produce large quantities of a single type of antibody and divide indefinitely. This technology allows for the consistent production of highly specific antibodies used in diagnostics, research, and treatment of diseases such as cancer, autoimmune disorders, and infectious diseases.
what is elisa
ELISA vs ELISPOT
Using soap as detergent to isolate proteins
What is polyacrylamide
Polyacrylamide is a synthetic polymer made from acrylamide subunits. It forms a gel matrix used in polyacrylamide gel electrophoresis (PAGE), a common laboratory technique for separating proteins or small DNA/RNA fragments based on their size and charge.
How it relates to gel electrophoresis:
In PAGE, a gel is made by polymerizing acrylamide with a cross-linker (usually bisacrylamide), forming a network with controlled pore sizes.
When an electric current is applied, molecules like proteins or nucleic acids migrate through the gel.
Smaller molecules move faster and farther through the pores, while larger molecules move slower—allowing for size-based separation.
Polyacrylamide gels are preferred over agarose gels for resolving small or closely sized molecules, especially proteins.
So, polyacrylamide is the material that forms the gel in PAGE, enabling precise separation of biological molecules.
Preparation of sample for loading onto SDS Page
SDS Page Western Blot Method
What is Western Blotting (huge overview)
Western blotting is a molecular biology technique used to detect specific proteins in a sample, based on their size and antibody recognition.
🎯 Purpose
- Identify presence or absence of a specific protein.
- Measure protein expression levels across different samples.
- Assess protein size (molecular weight).
- Study post-translational modifications, such as phosphorylation or cleavage.
Drawbacks of Western Blot
