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Western Blot - PowerPoint PPT Presentation


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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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Presentation Transcript
slide2

Steps:

1. SDS-PAGE

2. Transfer to membrane: "Blotting"

3. Detection of proteins

slide3

SDS-PAGE

SDS: sodium dodecyl sulfate

PAGE: polyacrylamid electrophoresis

slide7

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

slide9

Reductant:

DTT: Dithiothreitol

B-Mercaptoethanol

The reducing agent beaks any cystine (-S-S-) bonds formed between two cysteine residues

slide10

Other stuff in the sample buffer:

Glycerol

Bromphenolblue

slide13

Polymerization reaction: radical catalyzed reaction

http://www.davidson.edu/academic/biology/courses/Molbio/SDSPAGE/SDSPAGE.html

slide14

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

slide15

"Discontinuous" PAGE

Low pH (6.8)

Low ionic strength

Low Acrylamid concentration

FAST

High pH (8.8)

High ionic strength

High Acrylamid concentration

SLOW

slide17

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.