MCELLBI C110L Lecture Notes - Lecture 10: Primary And Secondary Antibodies, Agarose, Chemiluminescence
3 Dec 2020
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Lecture #10: agarose electrophoresis, kelp-based gelatin like substance, forms sieving mesh after heating/cooling. Ii. properties: requires, a crosslinker, a free radical initiator, a free radical stabilizer, can run under native or denaturing conditions, cheap, but unpolymerized gel is toxic, o2 quenches/slows polymerization, layer acrylamide with isopropanol or n-butanol after pouring. Some general considerations: gel follows laws of physics, p = iv. Increasing voltage increases heat load: gel can crack, stronger diffusion, lower resolution, proteins can denature at high heat (native gels, possible solutions: cool the gel, run at lower voltage. Reducing agents: prevent cysteines from cross-linking, prevent disulfide bonds, further removes secondary and tertiary structure, b-mercaptoethanol, dtt. Other notables: heat sample before loading, assures/accelerated unfolding in presence of sds, glycerol in the loading buffer. Increases density of solution to allow loading into wells: avoid concentrations of k, precipirates dodecyl sulfate can interfere with loading and running. Cl-not sieved, moves faster towards anode than proteins: 2.
