Western Blot

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Western Blot
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Steps 1. SDS-PAGE 2. Transfer to membrane
"Blotting" 3. Detection of proteins
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SDS-PAGE SDS sodium dodecyl sulfate PAGE
polyacrylamid electrophoresis
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The goal is to separate proteins according to
their sizes. How would you do that?
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(No Transcript)
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Remember
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SDS sodium dodecyl sulfate is a detergent (soap)
that can dissolve hydrophobic molecules but also
has a negative charge (sulfATE) attached to it
http//www.davidson.edu/academic/biology/courses/M
olbio/SDSPAGE/SDSPAGE.html
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www.ufs.ac.za
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Reductant DTT Dithiothreitol B-Mercaptoethanol
The reducing agent beaks any cystine (-S-S-)
bonds formed between two cysteine residues
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Other stuff in the sample buffer
Glycerol Bromphenolblue
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(No Transcript)
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How to make the gel?
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Polymerization reaction radical catalyzed
reaction
http//www.davidson.edu/academic/biology/courses/M
olbio/SDSPAGE/SDSPAGE.html
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Polymerization reaction
Catalysts APS Ammonium persulfate, radical
initiator TEMEDN, N, N', N'-tetramethylethylen
ediamine, free radical stabilizer
http//www.davidson.edu/academic/biology/courses/M
olbio/SDSPAGE/SDSPAGE.html
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"Discontinuous" PAGE
Low pH (6.8) Low ionic strength Low Acrylamid
concentration FAST
High pH (8.8) High ionic strength High Acrylamid
concentration SLOW
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Visualization of proteins on the gel Coomasie
stain
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OR do a western blot Proteins are transferred
to a protein binding membrane. We will use a
nitrocellulose membrane. Polyvinylidene
difluoride (PVDF) is also commonly used.
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OR do a western blot