SDS PAGE and Western Blot

Differences between agarose gel and SDS PAGE gel

Agarose: for DNA because larger pores

Polyacrylamide gel: for proteins because smaller pores

compare and contrast how we analyze DNA vs. proteins and why we have to use different methods

DNA - negative charge and linear

Proteins - must change 3D conformation of protein

• SDS: linearizes protein and gives it negative charge

• Reducing agent (B-mercaptoethanol): linearizes protein and breaks disulfide bonds

Proteins 1/6 size of DNA

average amino acid = 110 daltons

average nucleotide pair = 649 daltons

steps of SDS PAGE and reagents used

1) Combine Laemmli buffer with fish sample

• Tris buffer: sets pH

• SDS: linearizes protein and gives it negative charge

• Reducing agent (B-mercaptoethanol): linearizes protein and breaks disulfide bonds

• Glycerol

• Bromophenol blue

2) Incubate at room temperature

3) Heat block

• extract protein

• denature and linearize protein

4) Load samples onto gel

• charge gives direction

• separated based on size

• positive control: myosin light chain

5) Stain with Coomassie blue

• binds to arginine, histidine, lysine

Steps of a Western Blot

purpose: allow you to say that a particular band is a particular protein (proteins measured in kilodaltons rather than bp like DNA)

1) SDS PAGE to separate proteins based on size

2) Transfer proteins to a nitrocellulose membrane

• electroblotting

• prepare Western Blot Sandwich (filter paper, poly. gel, n. membrane, filter paper 2)

• no SDS b/c washed out thus proteins move from (-) to (+) by manipulating pH

• before transfer: p. gel has proteins

• after transfer: no protein on gel → moved to membrane

3) Probe the membrane with antibodies specific to a protein of interest

• Fc region: direct biological activity of antibody

• Fab region: antigen binding fragment

3a) block unbound membrane with milk (milk covers unoccupied spaces on nitrocellulose membrane and allows antibody 1 to bind specifically)

3b) add primary antibody (variable region recognizes myosin light chain)

3c) add secondary antibody (recognizes and binds to primary antibody)

• Horseradish peroxidase: cleaves 4CN to produce purple precipitate at location of the antigen (myosin)

3d) Substrate for colorimetric detection (4-chloro-1-naphthol)

antibody naming

(animal antibody was generated in) ___ anti- ___ (whatever the antibody recognizes)

ex: want to detect human CCR5 in a western blot

1) inject human antigen (CCR5) into mouse. then harvest mouse antibodies → mouse anti-human CCR5

2) inject mouse antibodies into a goat. then harvest goat antibodies

goat anti-mouse

primary antibodies recognize…

specific proteins

secondary antibodies recognize…

primary antibodies

T/F: Proteins are run on SDS PAGE gel in their native (folded) confirmation

False

What is purpose of reducing agent in the Laemmli sample?

linearize protein and break disulfide bonds

Why are we using polyacrylamide gel for sequencing proteins instead of agarose gel?

polyacrylamide gel has smaller pores and proteins are smaller than DNA

Which technique will be used to separate out the different STRs from each other after the PCR amplification?

Capillary gel electrophoresis

If trying to detect tropomyosin. Perform western blot and probe with a mouse antibody that detects and binds to tropomyosin. Which of the following antibodies would work as a secondary antibody for your experiment?

a) an antibody from goats that binds to mouse antibodies

b) an antibody from goats that detects tropomyosin

c) an antibody from rabbits that binds to mouse antibodies

d) an antibody from horses that detects tropomyosin

e) an antibody from goats that detects rabbit antibodies

a) an antibody from goats that binds to mouse antibodies

c) an antibody from rabbits that binds to mouse antibodies