Antigen Detection of Myosin Heavy Chain and Adipsin

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Antigen Detection of Myosin Heavy Chain and
Adipsin

• In myoblast/myotubes and fibroblast/adipocytes,
  respectively

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E63 3 day E63 7 day
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Antigen Detection

• We will be probing, with specific antibodies, the
  proteins spread out by size (from PAGE and
  western blotting) on last weeks nitrocellulose
  membrane
• Specifically probing for MHC (myosin heavy chain)
  and adipsin

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What is an antigen?

• Molecule that provokes the cellular production of
  specific neutralizing antibodies in an immune
  response
• Epitope a site on an antigen recognized by an
  antibody.

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Creating Polyclonal Antibodies
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Creating Monoclonal Antibodies

• Large quantities of a single type of antibody
  molecule can be obtained by fusing a B cell
  (taken from an animal injected with antigen A)
  with a tumor cells. The resulting hybrid cell
  divides indefinitely and secretes anti-A
  antibodies of a single (monoclonal) type.

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Using Antibodies for Microscope Detection
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Our Western Probing
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General Protocol

• First, block any available sites without protein
  with non-fat dry milk solution
• Treat the membrane with a primary antibody,
  specific for your target antigen (MHC or P16)
• Wash away any excess antibody
• Treat with secondary antibody that will
  specifically recognize your primary antibody.
• The secondary antibody is tagged so that it can
  be later visualized to detect the bound target
  protein.

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Buffers

• Blocking Buffer
• Reduces background binding to the nitrocellulose
  by the primary or secondary antibodies
• 5 Non-fat dry milk blocks unoccupied sites on
  membrane
• 1 Non-fat dry milk washes away unbound protein
• Tris Buffered Saline Tween (TBST)
• Tris buffered salts
• Keep pH, electrolytes, ions in correct balance
• important for proteins (antibodys)
  functionality
• Tween 20
• A detergent that reduces all molecules binding
  strength.
• Thus only the highest binding affinity bonds will
  form

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Our Primary Antibodies

• MF20
• Monoclonal antibody against MHC (myosin heavy
  chain)
• created by a hybridoma with a B lymphocyte,
  secreting IgG specific for MHC, and an
  immortalized mouse cell
• P-16
• Polyclonal antibody against adipsin
• Created by injecting goat with adipsin so it
  produces antibodies against adipsin. The
  subsequent IgG is isolated from blood.

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Secondary Antibodies
Mouse hybridoma cell line!

• Goat anti-mouse (for MHC)
• Made by isolating mouse serum IgG and injecting
  it into another mammal (ie. goat), which
  generates an immune response against the foreign
  protein (ie Mouse-IgG).
• These new antibodies are isolated from the goat
  serum and labeled with Alkaline Phosphatase (AP)
• Donkey anti-goat (for adipsin)
• Same as above but is made by isolating goat serum
  IgG and injecting it into a donkey

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Antibody Sandwich
myosin

• Advantages of doing this
• Increased Specificity
• Improved Signal

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Alkaline Phosphatase Reaction

• AP NBT BCIP
• Color reaction reagents
• The AP removes phosphates from BCIP (bromo
  chloro indol phenol), which can then combine with
  NBT (nitro blue tetrazolium) to form the purple
  color product formazan
• This reaction is performed in the dark because
  light energy could cause formazan deposit to take
  place at sites other than the AP-Ab-protein site.

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Blots
P16 against adipsin
MF20 against myosin
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Other colorimetric schemes

• HRP DAB (or cyanide)
• Horse radish peroxidase and di-amino benzidine
• Colloidal Gold
• Prohibitively expensive
• 10x more sensitive than APNBTBCIP
• Biotin-Avidin
• Have many APs attached
• Similar in cost to APNBTBCIP
• 5-20x more sensitive