IUB BIOL-L 211 - Molecular Biology Techniques I (3 pages)

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Molecular Biology Techniques I



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Molecular Biology Techniques I

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EMSA, DNase footprinting assay, and Chain-terminating method


Lecture number:
36
Pages:
3
Type:
Lecture Note
School:
Indiana University, Bloomington
Course:
Biol-L 211 - Molecular Biology
Edition:
1
Documents in this Packet

Unformatted text preview:

BIOL L211 Lecture 36 Outline of Last Lecture I Protein Purification Outline of Current Lecture I Techniques to examine DNA protein interactions II EMSA III DNase footprinting assay IV Chain terminating method Current Lecture Molecular Biology Techniques I Recall sequencing from prior lectures I Techniques to examine DNA protein interactions A Electrophoretic mobility shift assay EMSA B DNase footprinting assay C Chromatin immunoprecipitation ChIP D In Vitro experiments done outside of living organisms 1 Example PCR Polymerase chain reaction 2 Precise cellular conditions can never be perfectly replicated E In Vivo experiments performed in living organisms 1 Example clinical trials Drosophila studies glowing puppies II EMSA A Electrophoretic mobility shift assay 1 In vitro experimental procedure used to examine protein DNA interactions These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute 2 DNA probe radioactively labeled dsDNA fragment with a potential DNAbinding site 3 A purified protein of interest is mixed with the DNA probe 4 Gel electrophoresis is performed B Left lane Control reaction DNA is not mixed with the protein 1 Appears as a single band on the gel corresponding to free DNA only C Right lane Excess DNA mixed with protein of interest 1 Protein does NOT bind all of DNA DNA is in excess some free DNA remains 2 Protein DNA complex migrates slower in the gel 3 A shift in the location of the band in the right lane means that the protein has bound the DNA 4 No protein DNA interactions would yield only one band at the same location as that of just free DNA III DNase footprinting assay A Used to tell where specifically a protein binds on a strand of DNA 1 Many copies of a radioactive probe are produced 2 The protein of interest is purified 3 Control Reaction no protein is added 4 Experiment protein is mixed with probe 5 In both cases DNA is digested with DNase a



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