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Western Blot

Western Blot. Steps: 1. SDS-PAGE 2. Transfer to membrane: "Blotting" 3. Detection of proteins. SDS-PAGE SDS: sodium dodecyl sulfate PAGE: polyacrylamid electrophoresis. The goal is to separate proteins according to their sizes. How would you do that?. Remember.

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Western Blot

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  1. Western Blot

  2. Steps: 1. SDS-PAGE 2. Transfer to membrane: "Blotting" 3. Detection of proteins

  3. SDS-PAGE SDS: sodium dodecyl sulfate PAGE: polyacrylamid electrophoresis

  4. The goal is to separate proteins according to their sizes. How would you do that?

  5. Remember

  6. 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/Molbio/SDSPAGE/SDSPAGE.html

  7. www.ufs.ac.za

  8. Reductant: DTT: Dithiothreitol B-Mercaptoethanol The reducing agent beaks any cystine (-S-S-) bonds formed between two cysteine residues

  9. Other stuff in the sample buffer: Glycerol Bromphenolblue

  10. How to make the gel?

  11. Polymerization reaction: radical catalyzed reaction http://www.davidson.edu/academic/biology/courses/Molbio/SDSPAGE/SDSPAGE.html

  12. Polymerization reaction Catalysts: APS: Ammonium persulfate, radical initiator TEMED:N, N, N', N'-tetramethylethylenediamine, free radical stabilizer http://www.davidson.edu/academic/biology/courses/Molbio/SDSPAGE/SDSPAGE.html

  13. "Discontinuous" PAGE Low pH (6.8) Low ionic strength Low Acrylamid concentration FAST High pH (8.8) High ionic strength High Acrylamid concentration SLOW

  14. Visualization of proteins on the gel: Coomasie stain

  15. 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.

  16. OR do a western blot

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