Western Blot Steps 1 SDSPAGE 2 Transfer to
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 (sulf. ATE) attached to it http: //www. davidson. edu/academic/biology/courses/Molbio/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. html
Polymerization reaction Catalysts: APS: Ammonium persulfate, radical initiator TEMED: N, N, N'-tetramethylenediamine, free radical stabilizer http: //www. davidson. edu/academic/biology/courses/Molbio/SDSPAGE. html
"Discontinuous" PAGE Low p. H (6. 8) Low ionic strength Low Acrylamid concentration FAST High p. H (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
- Slides: 18