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

Western Blotting

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

Who is this person?

What did she discover?

And what does her discoveryhave to do with WesternBlots?

Two pathways of transposition:

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

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

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

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

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

Transposon-likemechanism of immunoglobulingene rearrangement

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

V and J Recombinationof Human IgG KappaLight Chains

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

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

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

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

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

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

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

Antibodies can bepolyclonal (producedby different B lympho-cytes; bind to different epitopes of the antigen), orantibodies can be monoclonal (made bya population of identical B cells; recognize a single epitopeof the antigen).

Pioneers in the developmentof monoclonal antibodies

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

Illustration of ELISA

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

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

Western or Immunoblot

“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

A cousin to the Western Blot in medicine

http://tools.invitrogen.com/content/sfs/manuals/pettopo_man.pdf

Why are we doing a Western Blot and what is the epitope on our protein that will be recognized by the Ab?

How is the experiment that we are doing similar toan ELISA or a traditional Western Blot?

How is it different?

Why did we choose to do the experiment the waywe are doing it?

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 (horseradish 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

What is HRP and what does it do?

Sigma PeroxidaseFrom Horseradish and SoybeanEC 1.11.1.7Synonyms: 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

http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/peroxidase-enzymes.html

What is HRP cont’d?

HRP, as the name implies, is an enzyme isolatedfrom 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 varietyof organic compounds, many of which have been developed for detection as colored compounds.

The color development system that we are using

V5adhP 6XHis

HRP

V5adhP 6XHis

HRP

SDS-PAGE Transfer Ponceau S Block

RinseAntibodyRinseColor Development

So let’s review:

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 EdwinSouthern and his invention.

http://www.oxfordtoday.ox.ac.uk/2005-06/v18n2/graphics/01big.jpg

−from “Fundamentals of Biochemistry” 3rd Ed. Voet, Voet, and Pratt (Wiley, 2008)

The Southern Blot

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

The Southern Blot in Forensic Medicine

And now there are other techniques…

Northern Blot – RNA analog of the Southern Blot

Eastern Blot – proteins from 2D SDS-PAGE transferredto 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