Free Essay

Western Blot

In: Science

Submitted By Unique797
Words 280
Pages 2
Antibodies and relatedness

Producing anti-cow antibodies

COW SERUM ALBUMIN

RABBIT ANTI-COW ALBUMIN ANTISERUM! (“COW ANTISERUM”)

CONTAINS

RABBIT ANTI-COW ALBUMIN ANTISERUM

ANTI-COW ALBUMIN ANTIBODIES

If we were to create anti-human albumin antibodies, why would they interact with serum albumin from animals other than humans?

ANTI-HUMAN ALBUMIN ANTIBODIES

mixed with serum albumin from..

HUMAN! CHIMP! GORILLA! BABOON STRONG! reaction HORSE! OX! DEER WEAK! reaction

CHICKEN! FROG NO! reaction

closely related to

distantly related to

somewhat related to

WESTERN BLOT An immunodiagnostic procedure used to detect (with extremely high specificity) particular proteins in a mixed solution, extract, or homogenate.
Native proteins are separated via gel electrophoresis, then transferred to a membrane where they are stained and hybridized with specific conjugated antibodies. Also called PROTEIN IMMUNOBLOT procedure.

General Procedure
1. Electrophoresis of sample.

2. Preparation of membrane/blotting of gel.

3. Staining of nitrocellulose blot.

4. Primar y antibody reaction.

5. Color development.

Preparation of Nitrocelluose Membrane

Nitrocellulose membrane

Separated Proteins Gelatin Primary antibody Secondary antibody (Color Development Solution)

HRP catalyzes a conformational change in chloronaphthol

PHLOEM

(e.g. chloronaphthol; TMB)

Fluorescent: secondary antibody conjugated with a light-emitting fluorochrome.

Enzymatic: secondary antibody conjugated with a enzymes such as HRP.

Other applications

Diagnostics: HIV Confirmatory Assay

HIV Confirmatory Assay

Disease progression: Chagas Disease!
Parasitic infectious disease of tropical America

Disease progression: Chagas Disease!
Parasitic infectious disease of tropical America

immune response in conjunctiva ! & facial region

damaged cardiac muscle

ACUTE

CHRONIC

Disease progression: Chagas Disease!
Parasitic infectious disease of tropical America

Anti-Trypanosoma IgM

Anti-Trypanosoma IgG

Silva, A.G. et al. (2004). Rev. Soc. Bras. Med. Trop. 37(6) !

!

(http:/ /dx.doi.org/10.1590/ S0037-86822004000600005)

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