We Can Raise Antibodies Against a Specific Antigen, How

We Can Raise Antibodies Against a Specific Antigen, How? BY loveyal 2345 midterm examination 2 Review Antibodies Experimental Purpose We can raise antibodies against a specific antigen (protein of intimacy) How? Polyclonal 1 antigen with many antibodies that bind to specific sites on the antigen (Received by injecting animal with protein of interest, waiting for that animal to build antibodies (B-lymphocytes). The lymphocytes be thence extracted which intermit us the polyclonal antibodies. Monoclonal I antibody that binds to a specific site on the antigen. (These are received by the same way as polyclonal, lodge you only extract ne antibody, and purport that into a cancer carrel to create a chimera of the two, the immortal cancer cell then acts like the monoclonal antibody. ) These are the best to use in experiments because they are specific to only ONE protein of interest. These antibodies can apply in experiments to scour a protein of interest Visualize a particular protein in a alert system or in a jelly How?? try out the gel to visualize where a protein is. Probing Protein Structure 1) X-ray crystallography cast off h your life producing sufficiently pure protein and obtaining a crystal protein (Crystallizing the proteins is a hard process) rack crystal protein with light, electrons, or radiation and examine the diffraction patterns with passing powerful computers -Analyze all the data while considering the amino-acid sequence and build a 3-D model of the protein. ) NMR-Nuclear Magnetic Resonance (Used rarely) For picayune proteins only Shoot concentrated pure proteins with strong magnetic field to generate hydrogen atom vibrations. Use computer program to measure reconstruct the body structure of the protein by measuring the hydrogen atom vibrations. Mass spectrometry is used as a precursor to both of these experiments. It generates the amino-acid sequence.Protein Purification 1) Grow Cells with protein of interest (transferred on plasmi d or native cell) 2) Lyse Cells -homogenization of tissuesdid in lab -cell lysis buffers dash cell membrane -sonicationsend sound waves through the cell to dissever membrane -pin- stack lysispush mixture through an extremely tiny hole (Force large molecules through a small opening causes them to break apart) 3) Centrifugation A) reparation Centrifugation B) Differential Centrifugation Sequential centrifugation increasing speeds (lowohigh) -low speed pellets = big things -high speed pellets= small things C) Velocity Centrifugation layer cell and lysate over a density slope and centrifuge to separate by density. Remove layers to separate proteins. D)Equilibrium Sedimentation other name for C 4) Column Cromatography 3 types Ion exchange (charge separation)protein adheres to beads of an polar charge Gel filtration (size separation)matrix has holes, the large proteins come out finis Affinity (Affinity separation)beads have something on it that only your protein binds to. ) Electrop horesis (small heap separation or detection) -use polyacrylimide gel (creates a mesh in the gel to separate proteins by size and charge. separates denatured proteins 6) Isoelectric focusing based on isolelectric point of protein2D electrophoresis Griffiths Experiment Conclusion heat killed bacterium transfigureed nonviolent bacteria Extract of heat killing S-strain transform R-strain to become S-strain Isolated transforming material (TM) and determined it was deoxyribonucleic acid not proteins that carried communicable information. (Took 1 5 years) How do we test?? Added proteases Injected into mouse lift should live (According to beliefs during that time period) Mouse however dies Added nucleases Mouse should die (According to beliefs during that time period) Mouse however livesThis illustrated that DNA carried the genetic information Hershey-chase Experiments Bacteriophagesvirus that infect bacteria Inject DNA into bacteria (naked)DNA unprotected by proteins Protein cause l eft outside of bacteria trail phages Label protein 7 groups of phages Label DNA in other groups of phages Mix both phage types with bacteria Blend bacterial mixture so that any viral move outside the cell are ripped off Pellet bacteria and get wind that only DNA label types is seen in pelleted bacteria Proved DNA carries genetic information 1) Grow bacteria with light DNA (14N) and doughy DNA (1 5N) which will separate to ifferent levels upon density-gradient centrifugation 2) Transfer heavy DNA and place in flask with light isotope Allows to eliminate conservative view 3) conflagrate DNA from step 2 to make it single stranded, then centrifuge.