We Can Raise Antibodies Against a Specific Antigen, How

We Can Raise Antibodies Against a Specific Antigen, How? BY loveyal 2345 midterm exam 2 Review Antibodies Experimental Purpose We flowerpot raise antibodies against a specific antigen (protein of interest) How? Polyclonal 1 antigen with many antibodies that bind to specific sites on the antigen (Received by injecting puppet with protein of interest, waiting for that animal to build antibodies (B-lymphocytes). The lymphocytes are then extracted which give us the polyclonal antibodies. Monoclonal I antibody that binds to a specific site on the antigen. (These are sure by the same way as polyclonal, expect you only extract ne antibody, and place that into a cancer cell 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 used in experiments to Purify a protein of interest Visualize a particular protein in a live system or in a gel HowProbe the gel to visualize where a protein is. Probing Protein Structure 1) X-ray crystallography Spend h your life producing sufficiently pure protein and obtaining a crystal protein (Crystallizing the proteins is a hard process) Shoot crystal protein with light, electrons, or radiation and examine the diffraction patterns with extremely powerful computers -Analyze all the data while considering the amino-acid sequence and build a three-D model of the protein. ) NMR-Nuclear Magnetic Resonance ( go ford rarely) For small proteins only Shoot concentrated pure proteins with strong magnetic field to generate hydrogen atom vibrations. Use computer program to measure reconstruct the structure of the protein by measuring the hydrogen atom vibrations. Mass spectrometry is used as a herald to both of these experiments. It generates the amino-acid sequence.Protein Purification 1) Grow Cells with protein of interest (transferred on plasmid or native cell) 2) Lyse Cells -h omogenization of tissuesdid in lab -cell lysis buffersbreak cell membrane -sonicationsend cash in ones chips waves through the cell to break membrane -pin-hole lysispush mixture through an extremely tiny hole (Force large molecules through a small outset causes them to break apart) 3) Centrifugation A) Regular 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 engrossment gradient and centrifuge to separate by density. Remove layers to separate proteins. D)Equilibrium Sedimentation another name for C 4) Column Cromatography 3 types Ion exchange (charge disengagement)protein adheres to beads of an diametrical charge Gel filtration (size separation)matrix has holes, the large proteins come out last Affinity (Affinity separation)beads have something on it that only your protein binds to. ) Electrophoresis (smal l volume 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 bacteria transformed nonviolent bacteria Extract of heat killing S-strain transform R-strain to become S-strain Isolated transforming cloth (TM) and determined it was deoxyribonucleic acid not proteins that carried genetic 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 Bacterio bacteriophagesvirus that infect bacteria Inject DNA into bacteria (naked)DNA susceptible by proteins Protein shell left outside of bac teria Label 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 parts outside the cell are ripped off Pellet bacteria and conserve that only DNA label types is seen in pelleted bacteria Proved DNA carries genetic information 1) Grow bacteria with light DNA (14N) and heavy 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) Heat DNA from stride 2 to make it single stranded, then centrifuge.