1. Biotechniques (BIOL 410)
Immunoblotting

2. Last time in Biotechniques…
Electrophoresis
Separate protein subunits by size
Or digested protein segments
Western blotting
Transfer protein subunits/segments to nitrocellulose

4. Immunoblotting Transferred proteins are not visible
Must stain to make them visable
Could stain in gel, like DNA agarose electrophoresis
Stain in nitrocellulose

5. Immunoblotting Staining on nitrocellulose is easier to observe
More stable (lasts loner)
Results are clearer
Use two sets of antibodies to stain

6. Immunoblotting Antibodies are target specific Bind to an antigen
Produced by immune system to target foreign cells or molecules
Mark for removal by Luckocytes

9. Immunoblotting Created by injecting foreign cells into host
Host produces antibodies
Immunoblotting uses two antibodies
First binds to the target (Antigen)
Second binds to first antibody, and includes a light sensitive tag.
Anti-antibody

13. Process Add first Antibody Wash to remove unbound antibody
Allow it to bind to the antigen (protein)
Wash to remove unbound antibody
Add second antibody
Allow to bind first antibody
Add HRP (Color Detection Reagent)

14. Using Data to Build a phylogenetic Tree
We can determine the relationship of fish using our data.
More closely related individuals should have more similar myosin/actin proteins

15. Measure protein distance
Start with ladder to create a standard line, similar to what we did for DNA
Measure proteins
Create a table to show how many protein subunits are of equivelent size between each species

18. Construct your tree Start with the two most closely related.
Then add each species that is most closely related to that group
Continue until all fish have been added to the chart

20. Today’s Lab Remove nitrocellulose from Blocker and stain for proteins
Antibody staining (Immunoblotting)
Observation and measurement of protein samples
Standard Cure, and Protein size
Phylogenetic analysis