L11-12 Analysis of proteins

Question 1

RNA and proteins

Answer 1

They are not directionaly proportional within a cell.

Question 2

Amino acid

Answer 2

contains carboxyl(COO-), amino (NH3+), R group

The R group can be hydrophilic-neutral, hydrophilic-charged,Hydrophobic.

Question 3

Levels of protein structure

Answer 3

Primary - A sequence of amino acids (polypeptide chain)

Secondary- Interaction between primary structures via H-bond, van der waals etc. which forms a complex structure
e.g alpha helix, beta sheets

Tertiary-Global protein

Interaction - H-bond, Hydrophobic effect, Disulfide bonds

Quaternary - Higher order assembly of proteins, multiple polypeptides, stabilized by non covalent interactions.

Question 4

Protein electrophoresis

Answer 4

Proteins have different charge and shape
To separate in electric field:
Must disrupt secondary and tertiary structures
Minimize aggregation between proteins in sample
Proteins in sample must have charge to migrate in same direction during electrophoresis

How to apply charge:
Sodium dodecyl sulfate (SDS)
It is an ionic detergent
Binds to proteins and disrupts non covalent interactions
Confers negative charge to denatured proteins
To remove di-sulfide bonds, Beta-mercaptoethanol is used.

NOTE: proteins move from negative to positive side

Question 5

SDS Polyacrylamide gel electrophoresis (PAGE)

Answer 5

It is made from an Acrylamide strands, which is cross linked by Bis-Acrylamide

Method:

• Sample is mixed with tracking dye
• Dye- Coomassie blue staining. Binds to protein not to gel. (quick+simple) 100ng accuracy
• Dye- SIlver staining. Based on reduction of silver ions bound to proteins. 0.1-10ng accuracy

Question 6

Protein detection by antibodies

Answer 6

Conjugating antiodies to enzymes or linking fluorescent molecule to antibodies.

Polyclonal body - Mixture of antibodies

Monoclonal body -

Question 7

Protein immunoblotting - Western blotting

Answer 7

Separate protein by SDS-PAGE

Electrotransfer of proteins into membrane

Proteins will remain in the same position

All gaps must be filled, otherwise antibodies will bind to the membrane

Antibodies will bind selectively to its intended target.

Antibody-enzyme conjugate is used to bind to primary antibody > add substrate for enzyme- produce color, fluorescence etc.

Western blotting application:
Estimate protein integrity
Estimate relative abundance of protein
Screen expression libraries

Question 8

ELISA

Answer 8

Very sensitive
High amount of samples processed
Quantitative technique (used for diagnostics etc.)

Question 9

Visualizing proteins in the cell

Answer 9

Immunomicroscopy.
VE-Cadherin stains the cell junction
DAPI is used to stain the cell nuclei

Question 10

Codons

Answer 10

3 nucleotides - codons

this allows 64 different kinds of amino acids

Question 11

Translational fusions and protein tags

Answer 11

Used is no antibodies are available
By using antibodies, a marker protein is binded to target protein to detect it.

Method:
Plasmid has a His-Tag, which is fused to protein via PCR.
The protein is detected by anti-his antibody by using Western blotting

Tags can be placed anywhere N terminal and C terminal

Question 12

Purification of His-tagged protein

Answer 12

Metal affinity chromatography.
Histidine binds strongly to Nickel bound to a matrix (membrane).

Additionally, competitor molecule, Imidazol releases His-tagged protein.

Question 13

In vivo detection (inside cell)

Answer 13

Translational fusions to auto fluorescent molecules.
Protein : GFP (Green fluorescent protein)
It defines where cell division occurs.

Question 14

Protein tags

Answer 14

Advantages:
DNA recombinant technology to add tag to protein of choice
Tag can be placed specifically
multiple tags can be placed on the same protein
Anti-bodies specifically binds/detects most of the tags available.
Allow purification of proteins

Disadvantages:
Need a cloned gene to create a translational fusion
May interfere with protein function
Tagged gene must be introduced into the cell or tissue of interest. (May be difficult to introduce to tissue of interest)

Question 15

Protein topology

Answer 15

Topology - what sort of conformation adopts when it is at the location it should be.

Where protein is localized what shape/form it takes.

Membrane protein topology - Proteins located in between the cell membrane. The part of the protein that sit in the membrane is called domain.

Question 16

Subcellular location reporter

Answer 16

A protein of its activity is influence its location (inside or outside the cell). Binds to proteins.
e.g PhoA - active only outside the cell, can detect by adding substrate, when degraded, color is produced.

Application:
Using PhoA to map where the proteins are located in terms of position relative to external and internal location.

Question 17

Proteomics

Answer 17

A group of techniques that look at proteome.
proteome - the entire complement of proteins that is or can be expressed by a cell, tissue, or organism.

2 Techniques (Method):
Protein electrophoresis
Mass spectroscopy

Technique : 2 dimentional electrophoresis

The charge on an amino acid is influenced by the change of pH hence each proteins will have a different isoelectric point.

First Dimention Isoelectric focusing
Seperates proteins according to their isoelectric point.
Proteins are denatured by urea and non-ionic detergent to dissociate charge (maintain unique isoelectricity)
Proteins are applies to a polyacrylamid gel strip containing a pH gradient
Protein is subjected to electric field proteins will migrate to their isoelectric pH. (no charge = no migration.)

2 Dimentional gel electrophoresis
Gel strip is treated with an SDS solution containing negative charge to proteins
Gel strip containing electrofocused proteins is placed horizontally on top of an SDS gel and subjected to electrophoresis
Proteins are separated according to size.

2D gel application:
Multiple proteins in 1 session
Identify disease or drug targets
Complement microarray experiments

Disadvantages:
Matching spots complex analysis a lot of subjective interpretation.
Many proteins are not expressed very well.
Very basic proteins or subjected to membrane do not separate very well, their isoelectric point passes a viable range, hence they are not represented well.

Then, identification: Peptide mass fingerprinting

Extract protein
Use Trypsin to cut lysine and argenine > fragments
Different size of fragments (pattern), their masses will be unique, hence can be fingerprinted by using mass spec.

Next > In silico (computer analysis)
Thousand of genome sequences available.
Genes are translated into proteins
Computer will use database to match the fingerprint of fragmented peptides.

Problems : identification can be more difficult if the protein undergoes post translational changes, hence the mass will be altered.