Molecular Biology: Blotting and Electrophoresis (MCB Lec 7)

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

detects protein - specific antibodies in protein

detect DNA; targets specific nucleotide sequences

detects mRNA - targets specific nucleotide sequences

labels specific AA or proteins in affinity-chromatography