NON-RADİOACTİV LABELS

S.VENKATESH

CONTENTS

Introduction Radiolabel Radioactive labels Disadvantages Non-Radioactive labelses Types Probes Hybridisation Detection Conclusion

Radiolabel is a part of Radioactivity to visualise components or target molecules in a biological system.

E.g.: P32, P33, S35

Introduction

Disadvantages:

Not easy to use radioactivity It is dangerous long exposure times and short half-life

limiting the stability of probes.

Non-Radioactive labels:

Fluorochromes Digoxigenin Biotin

Types.

Direct labeling The former includes direct incorporation

of fluorescent tags, or cross-linking enzyme molecules directly

to nucleic acid.

Indirect labeling involves the incorporation of nucleotides

tagged with a hapten such as biotin or digoxigenin (DIG) during

synthesis of the probe

Why DIG As a Signal Molecule?

DIGoxigenin is a steroid hapten from Digitalis lanata

A system for labeling nucleic acids and proteins Detection Options: color, fluorescence, chemiluminescence Faster, safer, and sensitive replacement for radioactivity

Detection

Used in standard blotting and hybridization procedures.

hybridize to a target sequence are detected with

streptavidin (biotin) or anti-DIG monoclonal antibody (DIG).

Enzyme activity can be detected either by a chemiluminescent

reaction whereby results are captured on X-ray film.

formation of a color precipitate deposited directly on the membrane.

Non-isotopic nucleic acid detection depends primarily on three variables:

1) the molecule or compound used to label the probe,

2) hybridization conditions, and 3) the detection method.

Probes

Definition: signal molecule that is used to identify a nucleic acid or protein of interest.

1. Two strand DNA probes 2. Synthetic oligonucleotide probes. 3. Single strand complementary DNA (cDNA) probes.

4. Complementary RNA (cRNA) probes.

Probes

Sense Probe Sense probe has the same sequence with the target nucleotide strand.

*Labeled nucleotid (UTP) *Labeled probe

Antisense Probe Antisense probe is used for the hybridization.

Hybridization Conditions

Once the probe is labeled and quantitated with an appropriate hapten, it can be hybridized to the target nucleic acid through complementary base pairing.

the complementary strands will re-associate or re-nature to form a double stranded structure .This process is called hybridization and refers to the

Detection Methods

Detection can be mediated either directly when using fluorescent haptens

or indirectly with the use of binding proteins like

antibodies or avidin/streptavidin as in the Detector system.

The specific antibody or binding protein is coupled to an enzyme or fluorochrome and subsequently visualized through a variety of signal-generating systems.

In enzymatic detection The enzyme reacts with a specific substrate to produce either a colored or luminescent product.

http://upload.wikimedia.org/wikipedia/commons/5/57/FISH_(Fluorescent_In_Situ_Hybridization).jpg

Detection of Hybridization

A)Detection of Non-radioactive Probes:

Immunohistochemical Method: A primary antibody against the label is used: For example; anti-digoxigenin. The next steps are the same with immunohistochemical method.

Biotin Avidin Method: If the probe is labeled with biotin, then avidin is used to visualise the complex.

Systems with direct signaling: Fluorochromes, Enzymes, and metals (colloidal gold).

B) Detection of Non-radioactive Probes :

Autoradiography:

1. Film Autoradiography : Hybridization slides are put on a specific rontgen film.This film must be sensitive to the radioactive label

used for hybridization. The film should be exposed for a certain

time. After developing the hybridization signal

is seen as dark areas on the film.

Detection of Hybridization

Film Autoradiography

B) Detection of Non-radioactive Probes :

2. Autoradiography on slides:

After hybridization the slides are covered with a photographic emulsion.

Exposed for a certain time.

After development the hybridization signal is seen as black dots on the section.

When analyzed with a dark field condenser the signal is seen as white shiny dots over a dark background.

Detection of Hybridization

GluR7

GluR7

GluR7

GluR6

GluR7 Exression in the Median Eminence

Concluson

In recent years, fluorescence, chemifluorescence and chemiluminescence have emerged as alternative technologies to the traditional radioisotope-based systems still widely used in many life science research applications.

For avoiding the problems of the radio active labeling the non radio active labeling is the wonder full technique and for the recent all gene manipulation techniques for identifying the target genes specially in the blotting techniques

References: Wilson.K & walker.j, Radioisotope techniques,

biochemistry and molecular biology, Cambridge university press (Cambridge university newyork), 2006, p: 621-655. 

Upadhyay.A,isotopes in biology, biophysical chemistry,Himalaya publishing house (Mumbai), 2003, 479-498.

 Brzofsky, J.A, Antigen and antibody interactions and monoclonal Antibodies, Fundamentals of Immunology, 3rd Edition, 1991.

 J. Osborn, Amersham Biosciences, A review of radioactive and non-

Radioactive-based techniques used in life science application, 2000.

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