MCB 253 Final Protein Characterization Native gel Principles behind the technique Non denaturing gel with no SDS Proteins moved based on size shape and charge not just size as in SDS Page Amino acid composition and post translational modifications will drive the protein to move a certain way depending on the pH of the buffer Can detect deamination aggregation binding events or other enzymatic activities Can recover proteins in their native state afterwards General protocols Can do same procedure as SDS PAGE but without SDS DTT and NO heat 1 2 3 4 Gel prep Mix sample with sample buffer Load into gel and run electrophoresis Stain with Coomassie Blue or perform Western Blot Expected outcomes Proteins move based on size shape and charge Various reagents and their purpose Acrylamide gel determines migration rate of proteins can use diff pore sizes Gel buffer has certain pH Sample buffer running buffer Staining solution Coomassie Blue to visualize bands SDS PAGE Principles behind the technique Determine size molecular weight of protein Can estimate number of polypeptides in your protein and the purity of your protein General protocols 1 2 3 4 5 6 Prepare gel Insert gel into chamber Mix protein with sample buffer SDS BME Heat sample Load into gel and run electrophoresis Stain with Coomassie Blue or perform Western Blot Expected outcomes Bands on the gel correspond to molecular weight size of your protein Various reagents and their purpose Polyacrylamide gel determines migration rate of proteins can use diff pore sizes TEMED catalyzes polyacrylamide gel polymerization o Accelerates the decomposition of persulfate molecules into sulfate free radicals and these in turn initiate the polymerization SDS sodium dodecyl sulfate denatures proteins to primary structure and gives them a uniform negative charge DTT breaks disulfide bonds removes the last bit of tertiary and quaternary structure by reducing disulfide bond BME breaks disulfide bonds disrupting tertiary and quaternary structure Sample buffer SDS dissolved in sample buffer Glycerol makes sample more dense than buffer so it stays in wells Western blot Principles behind the technique Separate proteins based on size and then identify the protein by using antibodies specific for your protein of interest antigen Transfer separated proteins from SDS PAGE gel onto nitrocellulose membrane Stain the membrane with antibodies Primary antibody binds to antigen Secondary antibody binds to primary antibody and will fluoresce substrate will react with enzyme on secondary antibody to generate a colored substance If primary antibody is mouse monoclonal antibody then secondary antibody must be an antimouse antibody General protocol 1 2 3 4 5 6 7 8 9 10 11 12 13 Carry out SDS PAGE Soak sponges and filter paper in blotting buffer Build transfer sandwich transfer gel to nitrocellulose membrane Put sandwich box in transfer chamber with blotting buffer Allow membrane to dry and store Cut membrane in two strips and soak in blocking buffer Wash in working buffer Incubate with primary antibody Wash with working buffer Incubate with secondary antibody Wash with working buffer HRP development Wash with water Expected outcomes Expect band to show up on nitrocellulose after incubating your protein with an antibody specific for your protein present Thickness of band corresponds to amount of protein Various reagents and their purpose 10 discontinuous PAGE gel determines migration rate of proteins can use diff pore sizes o The discontinuous buffer systems employ different buffer ions and pH in the gel and in the electrode reservoirs Samples are loaded onto a non restrictive large pore gel called a stacking gel which overlays a smaller pore resolving gel o Proteins concentrate or stack into narrow zones during migration through the largepore stacking gel and destack or resolve under the given conditions in the small pore resolving gel o Relatively large volumes of dilute protein samples can be applied to the gel BSA standard 2mg ml can be used as positive control prevents non specific binding of antibodies 1X electrode buffer with SDS SDS denatures proteins Sample buffer ME 10 SDS denatures proteins and gives them a uniform negative charge also breaks disulfide bonds Kaleidoscope protein weight marker Ladder to compare results to in order to find molecular weight and corresponding color HRP color development solution enzyme label used in conjugation with a secondary antibody that causes a color to appear Ponceau S Stain used to show the presence of proteins on the nitrocellulose membrane usually reversible Blotting Buffer necessary to transfer proteins from gel to nitrocellulose membrane Blocking Buffer Blocking is a very important step of western blotting as it prevents antibodies from binding to the membrane nonspecifically Working buffer minimized background and removes unbound antibody Tris HCl buffer protease inhibitor Fluorescent staining and microscopy Principles behind the technique Use dyes that are specific for different compartments of the cell nucleus plasma membrane or cytoplasm WGA Wheat Germ agglutinin makes plasma membrane fluoresce green binds to residues on plasma membrane o Member of the lectin family that binds to N acetyl D glucosamine and sialic acid residues found on the surface of cell membranes DAPI makes the nucleus fluoresce blue binds to minor groove of DNA o This particular stain binds to the A T rich regions in the DNA and when bound is more fluorescent Rhodamine Phalloidin makes the cytoplasm fluoresce red binds to actin General protocol 1 Wash cells with PB 2 Add glycine 3 Wash with PBS 3 times 4 5 6 7 8 9 10 Add Triton Wash with PBS 3 times leave PBS on slide after last wash Add dye WGA DAPI or RP to the PBS on the slide Wash with PBS 3 times Flip cells onto microscope slide Add mounting media Use clear nail polish to seal edges on slide Expected outcomes Nucleus should fluoresce blue if your protein localizes to nucleus Cytoplasm should fluoresce red if your protein localizes to cytoplasm Plasma membrane should fluoresce green if your protein localizes to membrane Various reagents and their purpose 0 15M Glycine quenches background images o Blocks unreacted aldehydes after fixation which can cause an increase in background fluorescence 0 4 Triton X 100 makes the cell membrane permeable to the dye 1X PBS to prevent background staining maintains cell at is physiological state buffer solution helps maintain pH Mounting media speeds up