SFP20 - Proteins as research tools Flashcards
What are the differences between polyclonal and monoclonal antibodies?
M are more expensive to produce. M are a single antibody species whereas P are a mixed population of antibodies. M only bind to a specifc site on the target molecule but the P antibodies can bind to different areas of the target molecule. P are tolerant to small chanegs of structure in the target protein whereas M antibodies may only recognise a specfic protein form.
What are the steps involved in polyclonal antibody production?
- Inject animal with antigen
- Antigen activates B cells
- B cells produce polyclonal antibodies
- Obtain antiserum from animal containing polyclonal antibodies
What is the purpose of SDS (sodium doceyl sulfate)?
It denatures the protein and prepares them for analysis
What does SDS-PAGE determine?
It separates proteins out based on their size
Why is it important that the proteins are denatured by SDS specifically before determining the size of them?
To break the bonds holding its 3D structure and to put them into linear polypeptide chains. This also coats them in equal negative charges.
How does SDS-PAGE work?
- Proteins are mixed with SDS and a buffer that helps to denature them and break apart any bonds holding their shape.
- A gel is made from a substance called polyacrylamide. The gel has tiny pores that act like a sieve to separate proteins based on their size.
- The prepared protein samples are loaded onto the gel, along with a marker of known protein sizes.
- An electric current is applied to the gel, causing the proteins to move through the gel. The SDS molecules bind to the proteins, making them negatively charged and separating them based on their size. Smaller proteins move faster, while larger proteins move slower.
- After the electrophoresis, the gel is stained to make the separated proteins visible. This allows researchers to see the different protein bands and estimate their sizes.
What is coomassie blue staining?
Coomassie Blue staining is a common method used to visualize proteins in polyacrylamide gels after separation by SDS-PAGE. It involves immersing the gel in a solution of Coomassie Blue dye, which binds to the proteins present in the gel.
What is western blotting used for after SDS-PAGE?
To detect specific proteins
How does western blotting work?
Transfer of the proteins onto a nitrocellulose membrane. Primary antibodies are then added which are specific to the protein of interest. A secondary antibody is added to the membrane which binds to the primary antibody if present. The secondary antibody is usually conjugated to an enzyme or a fluorescent dye.
How does ELISA (Enzyme-linked immunosorbent assay) work?
Sample (protein of interest) is coated onto a microtitire plate. Primary antibody specfic to the protein is added, then a secondary antibody conjugated to an enzyme is added. The substrate is added which will be catalysed to result in a signal. Then detect the signal.
If more of the protein is present, then more primary antibodies will bind, then more secondary antibodies, then a greater signal can be detected and the protein amount can be determined.
For immunohistochemistry and immunofluorescence, where are the antibodies binding to the proteins at?
Cells or tissue samples
Immunohistochemistry is quite similar to ELISA, same process
What is the difference with immunofluorescence?
There is a fluorescent tag present on the primary antibody. It is stimulated by a wavelength of light to show the location of the antibodies.
How does immunogold electron microscopy work?
Same principle as before but there is a gold particle conjugated to the antibody which is electron dense. This means that when it is under an electron microscope, it will be seen easily. Extremely high resolution for endogenous proteins.
