We Can Raise Antibodies Against a Specific Antigen, How

We Can countermand Antibodies Against a Specific Antigen, How? BY loveyal 2345 Midterm 2 Review Antibodies observational Purpose We can raise antibodies against a specific antigen (protein of interest) How? Polyclonal 1 antigen with galore(postnominal) antibodies that bind to specific sites on the antigen (Received by injecting brute with protein of interest, waiting for that animal to build antibodies (B-lymphocytes). The lymphocytes be then extracted which give 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, expect you only extract ne antibody, and place that into a cancer cell to create a chimaera 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 sensation protein of interest. These antibodies can used in experiments to Purify a protein of interest image a particular prot ein in a live system or in a gel HowProbe the gel to visualize where a protein is. investigate Protein Structure 1) X-ray crystallography Spend h your life producing sufficiently sharp 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 darn considering the amino-acid sequence and build a 3-D model of the protein. ) NMR-Nuclear Magnetic sonority (Used rarely) For niggling proteins only Shoot concentrated pure proteins with strong charismatic field to generate hydrogen instalment vibrations. Use computer platform to measure reconstruct the structure of the protein by measuring the hydrogen atom vibrations. Mass spectrometry is used as a precursor to twain of these experiments. It generates the amino-acid sequence.Protein Purification 1) Grow Cells with protein of interest (transferred on pl asmid or congenital cell) 2) Lyse Cells -homogenization of tissuesdid in lab -cell lysis buffersbreak cell membrane -sonicationsend expire waves by means of the cell to break membrane -pin-hole lysispush mixture through an extremely tiny hole (Force prodigious molecules through a small opening causes them to break apart) 3) Centrifugation A) Regular Centrifugation B) Differential Centrifugation Sequential centrifugation increasing rushs (lowohigh) -low speed pellets = big things -high speed pellets= small things C) Velocity Centrifugation layer cell and lysate over a density gradient and centrifuge to separate by density. call back layers to separate proteins. D)Equilibrium Sedimentation another name for C 4) Column Cromatography 3 types Ion exchange (charge separation)protein adheres to beads of an opposite 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 (small volume separation or detection) -use polyacrylimide gel (creates a mesh in the gel to separate proteins by size and charge. separates change proteins 6) Isoelectric focusing based on isolelectric point of protein2D electrophoresis Griffiths experimentation Conclusion heat killed bacterium transformed nonviolent bacteria extract of heat killing S-strain transform R-strain to become S-strain Isolated transforming material (TM) and unyielding it was deoxyribonucleic acid not proteins that carried genetic reading. (Took 1 5 years) How do we judge Added proteases Injected into mouse setback 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 protei ns Protein shell left outside of bacteria note phages Label protein 7 groups of phages Label DNA in other groups of phages strut both phage types with bacteria Blend bacterial mixture so that any viral parts outside the cell are ripped slay Pellet bacteria and observe 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) convey heavy DNA and place in flask with light isotope Allows to abolish conservative view 3) Heat DNA from step 2 to furbish up it single stranded, then centrifuge.