Western Blotting Lecture

1. What has been or will be done in lab this week:

2. Western Blotting
Who is this person?
What did she discover?
And what does her discovery
have to do with Western
Blots?
from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

3. Two pathways of transposition:
from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

4. from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

5. from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

6. from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

7. Transposon-like
mechanism of
immunoglobulin
gene rearrangement
from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

8. V and J Recombination
of Human IgG Kappa
Light Chains
from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

9. from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

10. from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

11. from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

12. from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

13. from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

14. from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

15. Antibodies can be
polyclonal (produced
by different B lympho-cytes;
bind to
different epitopes
of the antigen), or
antibodies can be
monoclonal (made by
a population of
identical B cells;
recognize a single epitope
of the antigen).
Pioneers in the development
of monoclonal antibodies
from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

16. Illustration of ELISA
from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

17. from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

18. Western or Immunoblot
from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

19. A cousin to the Western Blot in medicine
“The first step of a rapid strep test is the extraction of
specific Group A streptococcal carbohydrate antigen from
the swab. The swab is placed in a test tube and extracted.
The extract is applied to a nitrocellulose membrane
containing both immobil. Ab and nonimmobil. Ab to
different regions of the Group A strep antigen. The
nonimmobil. Ab are conjugated to dyed colloidal
gold particles. If Group A streptococcal carbohydrate
antigen is present in the extract, the conjugated Ab
bind to it, forming antigen-Ab complexes. These
migrate along the membrane until they reach the reaction
zone containing immobilized antibodies to the same Group
A strep antigen. These antibodies capture the antigen-
Ab complexes, forming a colored band or line
(usually pink or blue) in the reaction zone area.”
http://www.enotes.com/nursing-encyclopedia/rapid-streptococcus-antigen-tests

20. Why are we doing a Western Blot and what is the
epitope on our protein that will be recognized by the Ab?
http://tools.invitrogen.com/content/sfs/manuals/pettopo_man.pdf

21. How is the experiment that we are doing similar to
an ELISA or a traditional Western Blot?
How is it different?
Why did we choose to do the experiment the way
we are doing it?

22. The Anti-V5-HRP Antibody allows detection of recombinant
proteins containing the V5 epitope. This epitope is found in
the P and V proteinsof the paramyxovirus, SV5 (Southern
et al., 1991). The Anti-V5 Antibody recognizes the 14
amino acid sequence:
Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr
The Anti-V5-HRP Antibody is a mouse monoclonal IgG2a
antibody that is itself conjugated to an enzyme (horse
radish peroxidase, HRP). Therefore, we do not need to
use a 2° antibody, and we cut down on the time to
complete the experiment.
http://tools.invitrogen.com/content/sfs/manuals/antiV5_man.pdf

23. What is HRP and what does it do?
Sigma Peroxidase
From Horseradish and Soybean
EC 1.11.1.7
Synonyms: Hydrogen peroxide
oxidoreductase, HRP
Grinding extraction and initial filtration steps of HRP production
http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical
-enzymes/peroxidase-enzymes.html

24. What is HRP cont’d?
HRP, as the name implies, is an enzyme isolated
from the roots of the horseradish plant (Amoracia
rusticana).
HRP is a glycoprotein (18% carbohydrate) and
consists of single polypeptide with a catalytic heme
group. Its MW is 44,000.
HRP uses H2O2 to catalyze the oxidation of a variety
of organic compounds, many of which have been
developed for detection as colored compounds.
http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/
peroxidase-enzymes.html

25. The color development system that we are using

26. So let’s review:
SDS-PAGE Transfer Ponceau S Block
Color Development Rinse Antibody Rinse
HRP
V5
adhP 6XHis
HRP
V5
adhP 6XHis

27. How did the Western Blot get its name?
To answer that question, we have to go back a few
years to 1975 and talk about a guy named Edwin
Southern and his invention.
http://www.oxfordtoday.ox.ac.uk/2005-06/v18n2/graphics/01big.jpg

34. The Southern Blot
-from “Fundamentals of Biochemistry” 3rd Ed. Voet, Voet, and Pratt (Wiley, 2008)

35. The Southern Blot in Forensic Medicine
from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

36. And now there are other techniques…
Northern Blot – RNA analog of the Southern Blot
Eastern Blot – proteins from 2D SDS-PAGE transferred
to membrane and probed for post-translational modifications
Far-Western Blot – similar to Western but non-antibody
proteins are used as probes, so protein-protein interactions
Far-Eastern Blot – separation of lipids, blotting, and
probed for ligand binding or via enzymatic analysis