How does fluorescence microscopy work?
- First barrier filter only lets light of a certain wavelength range through
- Beam-splitting mirror reflects light below a certain wavelenth towards object, but transmits light from object above that wavelength
- Second barrier filter cuts out unwanted fluorescent signals –> emits light @ higher wavelength
**light emitted from second filter is at a diff wavelength then when it was transmitted through first filter
How does confocal laser scanning microscopy work?
- Laser shined through dichroic mirror
- Light reflects off 2nd dichroic mirror onto specimen stained w/ fluorescent dye
- Light reflects off specimen back through 2nd dichroic mirror
- Pinhole eliminates all the out-focused light and only allows some light through
What organism is green fluorescent protein isolated from?
Jellyfish
What is GFP used for?
GFP marker: can fuse DNA encoding GFP onto a gene encoding a protein of interest
The gene Src normally is activated by extracellular signals called growth factors. When the growth factors are present, Src instructs the cell to divide. A mutant form of Src is found in many cancer tumors and is active in the absence of growth factors. The normal Src is a:
Proto-oncogene
Describe the structure of an IgG antibody.
- 2 heavy chains (Y formation) connected to light chains (1 on either branch of the”Y”) via disulfide bond (3)
- 2 antigen binding sits on ends of light chian
What is the F(ab) fragment of an IgG antibody?
Variable region aka the binding site
What is the F(c) fragment of an IgG antibody?
Constant region that has exactly the same AA sequence for all the IgGs from that animal
How many F(ab) regions does an IgG antibody have?
2 b/c bivalent
How many F(c) regions does an IgG antibody have?
1
What does the F(ab) fragment bind to?
Small epitope (piece of an antigen) on the protein (antigen) of interest
Polyclonal vs. monoclonal antibodies?
- Polyclonal = multiple epitopes
- Monoclonal = highly specific, limited
What is an epitope?
Small piece (~6 AAs) on the surface of an antigen (whole protein) that is recognizable by an IgG
What is IgG?
Class of antibodies that is in its free form in the blood
What does each IgG consist of?
- 2 identical light chains
- 2 identical heavy chains
What is a western blot analysis used for?
To see if the protein of interest is present in your sample, how much of it is there, and what the molecular weight of the protein is
How does a western blot analysis work?
- Cells are lysed w/ SDS detergent
- Lysates subject to SDS-PAGE (gel electrophoresis) to separate all the proteins based on size (small = fast)
- Proteins are transferred along an electrical current from gel to a membrane
- Membrane is probed w/ an antibody specific to the antigen of interest
What is SDS? Function?
Strong ionic detergent used to solubilize membranes, denature proteins, and provide a uniform (-) charge
What kinds of proteins move fastest on the SDS PAGE?
Smaller proteins move faster and farther in the gel
Primary vs. Secondary antibody? Function of each?
- Primary: first antibody that recognizes your protein
- Secondary: recognizes Fc region of primary antibody –> amplifies signal
What property would you take advantage of to separate the ER from Golgi?
Density of the organelle
Antibodies (IgG) are powerful reagents because they bind to a specific antigen via the ___ and to protein A from bacteria via the ___.
- Fab region
- Fc region
