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18 Cards in this Set
- Front
- Back
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Why is it good that antibodies have very specific binding (in relation to protein detection)?
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good for isolation
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How are antibodies made?
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the B cells are activated by the antigen and make the antibody
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Why do antibodies display highly variable regions?
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for binding diversity
specificity comes from variable regions |
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Antibody Affinity Chromatography
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start with pH 7 buffer which allows the charges of the antibody antigens to interact.
Elute with acidic pH 3 buffer which increases the H+ so it binds to carbonyl Coo- -you can cover up the charge and the antigen is released |
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Differential Centrifugation
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particles separate on the basis of mass
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Rate-Zonal Centrifugation
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use density gradient to separate on basis of mass
used to separate particles whose masses are close |
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How do you denature proteins?
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treat with 8M urea and Beta mercaptoethanol
used with gels proteins re-nature upon dialysis |
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What does SDS (sodium dodecyl sulphate) do?
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make the proteins negative
used in electrophoresis |
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What does SDS gel separate on the basis of?
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MW
length of gel = R amount it moves = D |
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Gel Filtration Chromatography
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"hole" filtration
big and small proteins with balls that have holes. only the small proteins can fit through the holes and get stuck in gels. Large proteins are moved out of the tube. |
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Ion exchange chromatography
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based on charge
beads of one charge attract proteins of opposite charge elute proteins with salt solution |
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Affinity Chromatography
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bind a ligand to the bead
ligand binding proteins seek ligands on the bead elute the ligand binding proteins ex. load actin and vilin onto beads coated with DNAse 1 in presence of Ca. Both proteins form a complex with DNAse 1. Elute using Ca free buffer. Good for cytoskeleton proteins actin and villan |
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Isoelectric Focussing deals with what?
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2D gels
makes a pH gradient and proteins are separated by isoelectric point |
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Western Blotting
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electrophoresing proteins, blotting them to a membrane and reacting them with a specific antibody or antiserum.
antibody is detected with a labeled secondary antibody or protein A. (transfer image from gel to porous paper) used only for proteins (not nucleic acids) |
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What are radioactive isotopes used for?
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to locate molecules in cells and gels
low energy = better resolution |
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Edman Degradation
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determines the amino acid sequence by labeling the N terminal with phenyl-isothiocyanate (PITC)
pulls off one amino acid at a time detects the type of amino acid by liquid chrom. |
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Mass Spectroscopy
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ionizes molecules and shoots them towards a target
times of flight are related to their masses (longer in flight, the bigger you are) masses tell you about the composition (nature) of the molecule used once the polymers are already separated qV is the energy you get from the electric field |
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Solid Phase Peptide synthesis
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by condensation reactions
aa1 attached to bead Aa1 blocked by tBOC (tetrabutyloxycarbonynl) block removed by trifluoroacetic acid tBOC blocked aa is added by coupling agent dicyclohexylcarbodiimide |
