Western Blot

Question 1

Electrophoresis

Answer 1

transfers from gel to nylon membrane

Use electrical charge to take out of gel and adhere to pos charged membrane

Question 2

Anode

Answer 2

Positive electrode

Toward which - charged prot will migrate

Question 3

Cathode

Answer 3

Neg electrode

Need dilute acetic acid for buffer

Question 4

NBT/BCIP

Answer 4

Nitro blue tetrazolium
Chromogenic substrate
Yields insoluble colored product

Question 5

Alkaline-phosphatase conjugated streptavidin

Answer 5

Detects all biotin-labeled prot

Question 6

Alkaline-phosphatase conjugated goat anti-mouse IgG

Answer 6

Detects only mouse IgG

Question 7

Alkaline-phosphatse conjugate goat antibody to unrelated prot

Answer 7

Control to detect prot that non specifically bind goat Ig regardless of Ag specificity

Question 8

Biotin

Answer 8

244 Da
Carboxylic acid found in enzymes and a B vit
Binds to avidin (egg whites) and Streptavidin
Used to label cells

Question 9

Streptavidin

Answer 9

66kDa
From bac Streptomyces avidinii
Non-glycosylated 
Neutral at pH 7
Less non-specific binding
Biotin-Streptavidin is strongest non-covalent binding known (Kd=10^15M)
Tetrameric prot can bind 4 biotin
Used conj to Alkaline phosphatase to detect biotin labeled prot