Western Blot.

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Presentation transcript:

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

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

www.ufs.ac.za

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

Other stuff in the sample buffer: Glycerol Bromphenolblue

How to make the gel?

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

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

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

Visualization of proteins on the gel: Coomasie stain

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.

OR do a western blot