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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2. Transfer to membrane: "Blotting"
3. Detection of proteins
SDS: sodium dodecyl sulfate
PAGE: polyacrylamid electrophoresis
How would you do that?
SDS: sodium dodecyl sulfate is a detergent (soap) that can dissolve hydrophobic molecules but also has a negative charge (sulfATE) attached to it
The reducing agent beaks any cystine (-S-S-) bonds formed between two cysteine residues
APS: Ammonium persulfate, radical initiator
TEMED:N, N, N', N'-tetramethylethylenediamine, free radical stabilizer
Low pH (6.8)
Low ionic strength
Low Acrylamid concentration
High pH (8.8)
High ionic strength
High Acrylamid concentration
Proteins are transferred to a protein binding membrane.
We will use a nitrocellulose membrane.
Polyvinylidene difluoride (PVDF) is also commonly used.