UNIT 1 MCB FINAL (part 2)

Fractionation based on charge
Anion exchangers: positively charged resin beads attach to negatively charged
molecules
Cation exchangers: negatively charged resin beads attach to positively charged
molecules

Based on size
larger move more rapidly
estimates MW
porous beads attach to small molecules

based on binding properties
substrate binds to enzyme
good for isolating proteins
examples: DNA, antibodies and antigen

based on polarity
non polar elutes first
bonds with polar molecules

clone and manipulate genes
add affinity tags to proteins of interest

based on charge and size
molecules go to cathode if positive and anode if negative
different gels for different solvents

Polysaccharide agarose --> separates nucleic acids
Cross-linked polyacrylamide --> separates proteins

Native gels
SDS page
Isoelectric focusing
2D gel

type of electrophoresis; proteins are separated by native site

proteins are separated by size
adding SDS make protein negative proprtional to its mass
SDS causes denaturation
proteins are put away from positive charge
small proteins travel farther

gel is pH gradient and proteins separated by charge
when pH of gel is = to pI of protein, protein will not move
negative charge moves to positive (low pH) vice versa

SDS page + isoelectric
separate by weight and charge

used to detect certain substances in sample
SNOW DROP acronym

labels specific AA or proteins in affinity-chromatography

- Actin filament
- Intermediate
- Microtubules

- organizes and supports cytoplasm
- guides intracellular traffic (mostly Microtubules)
- Pulling apart of chromosomes in mitosis (microtubules)
- muscle cell contraction

- thinnest
- highly concentrated in the cortex
- supports shape of plasma membrane

- G-Actin
- F-Actin
- Polymerization

Associate to form polymer with subunits added to both ends
- subunits add more rapidly to + end
- shrinking occurs in - end
Filmentous form

process to create polymers
- reversible
- constantly grows and shrinks
growth rate is equal to shrink rate

- largest of the 3
- POLYMERS of tubulin (dimer of alpha and beta subunits).
- bind to G nucleotide

inaccessible and always stays as GTP

more exposed to the solvent and can be hydrolyzed from GTP to GDP

2 a-helixes forming a left handed coil, ideal version is Heptad Repeat (Leucine residues, in position 4, every 7 away)
Every 3.6 residues make a turn and hydrophobic interactions coil them together

- Crucial for extracellular matrix and connective tissue,
- 3 polypeptide chains (triple-helix),
- every 3rd residue is glycine
glycine --> flexible = collagen

very high temperature cause denaturation of proteins, lower enzyme activity
lot of enzymes require the catalytic residues to be in correct ionization state, if not can impact enzyme activity (changes in pH)

a complete catalytically active enzyme together bound with its coenzyme and/or metal ions

the protein part of the holoenzyme

Binding: binds to substrate
Positioning: Binding places the sugar bond to be split near two key lysozyme residues, Glu35
and Asp52.
Activation: ASP 52 acts as a nucleophile and forms a temp bond with sugar (covalent catalysis)
Involvement of water: : Glu35, now negatively charged, makes water a better nucleophile.
Water's OH- joins the sugar's carbon, and H+ joins Glu35 (general base catalysis).
5. Hydrolysis: The sugar bond between sugar units D and E is hydrolyzed, and the enzyme returns
to its original state.