10. Autoradiography. X-ray Crystallography

Question 1

What is microscopic autoradiography? and what molecules does it localise?

Answer 1

MICROSCOPIC AUTORADIOGRAPHY:

METHOD OF LOCALIZING NEWLY SYNTHESIZED MACROMOLECULES (a molecule containing a very large number of atoms) SUCH AS:
•DNA
•RNA
•PROTEINS
•GLYCOPROTEINS
•POLYSACCHARIDES

IN CELLS/TISSUE SECTIONS

Question 2

What are metabolites?

Answer 2

METABOLITES:

SUBSTANCES FORMED IN/NECESSARY FOR METABOLISM

Question 3

What does autoradiography use to generate an image?

Answer 3

Autoradiography uses radioactivity to generate an image of the cell or tissue

Question 4

How can you introduce a radioactive substance in AR?

Answer 4

Can introduce a radioactive substance by:

• metabolic pathway
• bound to a receptor
• enzyme
• hybridized to a nucleic acid

By introducing a radioactive subs you can use the decay of the substance create an Autoradio-graph image

Question 5

Examples of Radioactively labelled metabolites?

Answer 5

• Nucleotides
• Amino acids
• Sugars

Question 6

Process of AR?

Answer 6

-Macromolecules w/ RLM emit weak radiation – the radiolabelled sections are limited to specific regions
-The radioactive substance binds to the selected parts of the cell depending on how introduction occurred.
-Slides w/ radiolabelled cells/tissue sections coated in a darkroom w/ photographic emulsion
-The emulsion contains:
•Silver bromide crystals
•The SBC act as micro detectors
•They respond to radiation the same way they respond to light in photographic films
-After exposure to lightproof boxes – slides are developed photographically
-SBC reduced by radiation produce small black grains of metallic silver – under TEM/light microscope, it shows locations of radiolabelled macromolecules in tissue

Question 7

What is XRC? What can you identify with this?

Answer 7

Allows us to identify the atom/molecular structure of crystal based on the diffraction of x-rays when shone onto crystals

X-RAY CRYSTALLOGRAPHY:

TOOL USED TO IDENTIFY ATOMIC/MOLECULAR STRUCTURE OF A CRYS-TAL. IN WHICH THE CRYSTALLINE ATOMS FORM A BEAN OF INCIDENT X-RAYS TO DIFFRACT IN MANY SPECIFIC DIRECTIONS.

Question 8

How can you prod a 3D image in XRC?

Answer 8

A crystallographer can produce 3D pictures of the density of e- within the crystal by:

• Measuring angles of diffracted beams
• Measuring intensities of diffracted beams

Question 9

What can e- density tell us in XRC?

Answer 9

The e- density can:

• Determine mean positions of atoms in crystal
• Their chemical bonds
• Their entropy

Question 10

What can XRC work for?

Answer 10

The mechanism can work for biological molecules:

• Vitamins
• Drugs
• Proteins
• DNA