MCB 2210 001 1 26 2015 Epifluorescence Microscopy improves contrast and allows specific structures to be labeled o Shine a light from outside onto specimens using mirrors and filters Filters only let certain wavelengths of light through Only see light emitted by specimen increases the contrast o Fluorescence a molecule absorbs light of one wavelength and them re emits it as a longer wavelength Absorbs a higher energy wavelength and emits a lower energy one o Fluorophores fluors bind to and label specific structures that they are targeted at There are a variety of different types What you see in the image is what the fluor targets directly Antibodies can be used because they bind to specific proteins To make an antibody o Obtain a pure protein of interest and inject it into an animal o The animal s body recognizes it as foreign and mounts and immune response o Individual B cells each produce a unique antibody that recognizes a specific amino acid sequence called the epitope Monoclonal antibodies made by cloning a single B cell antibody producer that recognizes target epitope o All molecules produced are identical Polyclonal antibodies mix of different antibodies produced by host animal s immune response against various epitopes of the target protein o Isolated from blood Immunocytochemistry uses antibodies to visualize specific proteins o To label structures that are inside of the cell they must be Fixed add chemicals to cross link them to nearby cells Freezes cell structure in place so that it doesn t die Permeabilize add a detergent to perforate the membrane so the antibodies synthesized can be added o Primary antibody the antibody that s been isolated and added to the cell o Directly labeled directly add fluor to target protein o Indirectly labeled use a secondary antibody an antibody against antibodies to bind the primary antibody Marker coupled directed o Can t be done on live cells because it kills the cell except for an extracellular protein Immunoblotting Western Blotting determines the size of protein of interest via electrophoresis o Add SDS to the polyacrylamide gel it s a detergent that denatures unfolds the proteins and gives the a negative charge Separates them by size via attraction between the positive and negative charge electric current Small molecules will move farther faster o To detect the antibody First add the primary antibody and then the secondary antibody o React with the substrate for the secondary antibodylinked enzyme to detect the protein of interest Immunoisolation Immunoprecipitation takes advantage of the fact that a bacterial protein protein A has a high affinity for antibodies o Grind up cells and incubate with the antibody that recognizes target protein o Add protein A beads o Then isolate the beads they have become coated with the antibodies and then attract the protein of interest Fluorescent proteins are genetically encoded fluorescent markers that can be fused to proteins of interest at the DNA sequence level Can be used with live imaging GFP green fluorescent proteins Gene for GFP is taken and put into a plasmid with a strong usually viral promoter that s upstream to it Transfer the plasmid to the target cells get them across the membranes and to the nucleus where they can be transcribed and expressed Tissue specific promoters can be used to visualize proteins in specific tissues Transfection infect a cell with foreign DNA to cause a foreign protein to be expressed in a cell o You can express Molecules that aren t normally present in a cell Mutant molecules that are constitutively active active all the time Mutant molecules that are dominant negative don t function and block the function of the cell s own version of the molecule Prevent protein function o Can be transient temporary or stable permanent Transient expression is directly from the plasmid which eventually wears off Stable the plasmid genes become integrated into the DNA genome and had the potential to become heritable o Protein expression can be general in all tissues or under the control of a tissue specific promoter Small interfering RNA siRNA knock out specific proteins of interest in cells o shRNA short hairpin RNA is mixed with a virus o The cells get rid of shRNA gained by infection The genes leave as siRNA RISC complex is added which unwinds siRNA It s associated with target mRNA complimentary The targeted mRNA is cleaved Engineering siRNA and transfecting cells leads to single stranded RNA that compliments mRNA and destroys it
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