Lecture 6

How can we identify the unknown proteins?

• Western Blotting

  • Transfer the proteins onto a nitrocellulose membrane

  • Use antibodies to identify specific proteins

How to make antibodies

• Purify your protein

• Inject into an animal (rabbit or goat)

• Bleed animal after month or so

• Purify antibody from the blood

Staining of the Blots

• Protein stain

  • No different from the stained PAGE Gel

• Use antibodies to identify specific proteins

• But wont the antibodies stick non-specifically to the nitrocellulose?

  • Incubate blot with Bovine Serum Albumin

  • Now no other proteins can stick to the blot

Stain” Blot with the Primary Antibody

• incubate with rabbit anti-mouse “C” antibody

  • Use pre-Stained Standard Proteins

  • Specific antibody binds only to C

antibodies are invisible, What are our options

1. Directly Label the antibody

• Fluorescent compound

• Radioactive compound

• Bind an enzyme to the antibody)

2. Use a labeled Secondary antibody

• Enzyme Conjugated Goat anti-rabbit Immunoglobulin antibody
  • Immunoglobulin = generic name for the antibody protein
  • Antibody = used for specific antibodies

Labeled Secondary Antibody binds to the Primary Antibody

• Primary Antibody (rabbit anti-mouse C protein Binds to the specific C protein)

• Secondary Antibody (enzyme labeled goat anti-rabbit Ig Binds to the Primary Antibody)

• Enzyme linked to the Secondary Antibody

Secondary Antibodies

• must be from a different species than the primary antibody

• Primary Ab is Goat anti-???
  • Secondary must be from some animal other than a goat!
  • Rabbit anti-goat Ig
  • Mouse anti-goat Ig

• If primary antibody = mouse anti-phospho-tyrosine
  • Secondary antibody must be goat, rabbit, donkey, etc. (anti-mouse Immunoglobulin)

What Enzyme to use to label the secondary antibody?

• Inexpensive

• Easy to make

• Has substrates that form insoluble, colored precipitates
  • Horseradish Peroxidase (HRP)
  • Alkaline Phosphatase (AP) – We will use this enzyme

• Substrate?
  • BCIP (5-bromo-4chloro-3-indolyl phosphate)
  • Acridan Based substrate (CDP-Star in this case)

Degradation of the Substrate

• Alkaline Phosphatase (AP) hydrolyzes CDP-Star to remove a phosphate

• The intermediate dioxetane phenolate anion is formed and once it decomposes light is emitted at 475 nm

Result: Only the “C” Band stains

• We can visualize the “C” protein band

• Estimate the size of the “C” protein as about 55 kDa

  • This “C” protein is NOT carbonic anhydrase!

Staining of the blots

• EGF binding to the EGF-receptor
  • Activates the EGF-R which is a Kinase
  • EGF-R phosphorylates itself and other proteins at tyrosine residues
  • This sets off a kinase tyrosine phosphorylation cascade signaling pathway

Stain for Tyrosine phosphorylated proteins

• EGF stimulation should result in the tyrosine phosphorylation of several proteins
  • EGFR activates other kinases
  • These kinases activate other kinases
  • Kinase cascade

• Shows that the pathway has been activated

• What We Expect to See:

  • Controls compared to EGF Treated:
    1. More bands
    2. Some heavier stained
    3. Some bands in the EGF treated but not in the control