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?

6. Remember

7. SDS: sodium dodecyl sulfate is a detergent (soap) that can dissolve hydrophobic molecules but also has a negative charge (sulfATE) attached to it

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

10. Other stuff in the sample buffer:
Glycerol
Bromphenolblue

12. How to make the gel?

13. Polymerization reaction: radical catalyzed reaction

14. Polymerization reaction
Catalysts:
APS: Ammonium persulfate, radical initiator
TEMED:N, N, N', N'-tetramethylethylenediamine, free radical stabilizer

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

16. Visualization of proteins on the gel:
Coomasie stain

17. 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.

18. OR do a western blot