BIOL300%20Lecture%202%20S.H.

BIOL300%20Lecture%202%20S.H. - Lecture 2 General principles...

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Unformatted text preview: Lecture 2 General principles II: Ligand- receptor binding and molecular analysis of signaling proteins Reading for Lecture 2: Lodish Ch. 15, pp. 627-631; Ch. 3, p. 91 Adds negative charge to proteins SDS Cut out bands, renature and test for activity; digest active band with trypsin (protease that cuts after lysine and arginine). Do mass spectrometry Mass spectrometry Tryptic peptides are ionized by laser and travel in electric field to detector. Time of flight is inversely proportional to mass. Peptide mass fingerprint can be compared to database of all proteins in an organism Purifying the receptor or ligand Ligand- Ligand-Receptor binding The more ligand OR receptor, the more RL When half the receptors are occupied then [L]=K d , [L] = free ligand concentration [R] = free receptor concentration The dissociation constant K d measures the affinity of the receptor for the ligand k off k on = First measure total binding, then nonspecific binding after saturation with unlabeled insulin, then calculate specific binding Binding assay to measure K d Kd gives the approximate concentration at which ligand is active First measure total binding, then nonspecific binding after saturation with unlabeled insulin, then calculate specific binding Binding assay to measure K d •An antagonist can block the binding of the natural hormone...
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This note was uploaded on 10/12/2011 for the course BIOL 300 taught by Professor Nilson during the Spring '11 term at McGill.

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BIOL300%20Lecture%202%20S.H. - Lecture 2 General principles...

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