In 1949, a team led by chemist Linus Pauling placed hemoglobin solutions from people with a disabling form of anemia and from healthy volunteers in an electric field, and found that the two samples migrated at different rates. In this way, the technique of electrophoresis helped decipher the molecular abnormality behind sickle cell disease

Separation of NA and proteins according to size, based on movement through a gel medium when an electric field is applied.

Separation of serum protein, enzymes, hemoglobin's, lipids,

migration of charged molecules in solution in response to an electric field.

Their rate of migration depends on the strength of the field; on the nett charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving.

Various types – defined by support used Paper – amino acids, small peptides Polyacrylamide – Proteins, small DNA/RNA (<500bp) Agarose – DNA/RNA

 is a cross-linked polymer of acrylamide. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%  is used in separation of Proteins, small DNA/RNA  Polyacrylamide is in liquid form at high temperature and gel for (semisolid) low or normal temperature Please check the gel getting hard by the addition of bisacrylamide that forms cross linkage with the acrylamide

 Increase concentration of the gel will decrease pores → high selectivity  The way of spile the gel in the plate is vertical  Use spacer to separate the plates  after adding the gel between the two plates cool the gel  Add the gel at power supply and connect the cathode and the anode

 Remove spacer and add buffer  Add the proteins (using the gel well)  Open the electric field  Protein with different molecular weight will be separated  The movement of proteins will be to the (+)

 during the movement of proteins you cant know when the protein reach the edge of gel  For that reason you should use marker molecule  Example for marker molecule (bromophenolblue)

 you should use protein stain  Examples for protein stains Color Silver Stain0.1 ng protein per band Krypton Fluorescent Stain 2 ng protein per band Imperial Stain (Coomassie R-250)6 ng protein per band GelCode Blue Stain (Coomassie R-250)10 ng protein per band

 Proteins are amphoteric compounds  In a solution with a pH above its isoelectric point, a protein has a nett negative charge and migrates towards the anode  Below its isoelectric point, the protein is positively charged and migrates towards the cathode.  At a given pH therefore, and under non- denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules.

 denatures the proteins by heating I  Stabilized the denaturation by SDS  SDS is Sodium dodecyl sulphate  is an anionic detergent which denatures proteins by "wrapping around" the polypeptide backbone  and SDS binds to proteins fairly specifically in a mass ratio of 1.4:1.  SDS confers a negative charge to the polypeptide in proportion to its length

SDS: Hydrophobic Hydrophilic Folded protein SDS  -mercaptoethanol Heat

 I can make all proteins with negative charge by adding mercaptoethanol OR dithiothreitol

 Intensity of the color is Proportional to the Molecular weight Please check it is the concentration

This is valid for agarose gel, so it is better to move this slide forward

 is a polysaccharide extracted from seaweed. It is typically used at concentrations of 0.5 to 2%. The higher the agarose concentration the "stiffer" the gel  Used to separate DNA/RNA.  Its horizintal  Repaet the Polyacrylamide process

 Dye  Audioradiography 32 P,  Blotting

ethidium bromide ( highly carcinogenic, binds DNA and fluoresces under illumination with UV light ) cyper green ( not carcinogenic, binds DNA and fluoresces under illumination with UV light )  both of them will not gives color until binding to DNA  Intinsity of the color proportional with molecular weight Be sure

 Blotting – Transfer of DNA, RNA or Proteins, typically from a electrophoresis gel to a membrane e.g. nitrocellulose. This membrane can then be subject to further techniques such as hybridization.  Hybridization – Process where two complementary single strands of nucleic acid (DNA or RNA) form a double helix. This slide and the next are not relevant and can be removed form the presentation

 Using specific probes that are labelled specific sequences of DNA can be identified.  There are three main hybridization techniques which vary in the sample blotted and the probes used; 1. Northern Blot-Transfer of an RNA sample separated and identified using DNA or RNA probes. 2. Southern Blot-Transfer of an DNA sample separated and identified using DNA or RNA probes. 3. Western Blot- Transfer of an Protein sample separated and identified typically using an antibody. x

 A ladder is a mixture of DNA fragments of selected sizes  When run in a gel electrophoresis, these fragments will separate into distinct bands that can be used as references

Reference Ladder An important point to remember is that the intensity of the band is proportional to the amount of DNA found in the band This is OK, but previously U said that the intensity of the band is proportional to its molecular weight

Polyacrylamideagarose significantly more annoying to prepare than agarose gels Agarose gels are extremely easy to prepare Acrylamide is a potent neurotoxinIt is also non-toxic small range of separation, but very high resolving power range of separation, but relatively low resolving power For proteinsFor NA

 remain negative at any pH used for electrophoresis  and in addition carry a fixed negative charge per unit length of molecule, provided by the PO4 group of each nucleotide of the the nucleic acid.  Electrophoretic separation of nucleic acids therefore is strictly according to size.

Denaturing For DNA, RNA by urea

 Two-dimensional electrophoresis Insert here the 2D animation that I gave to U Depend on isoelectric focusing and molecular weight of the proteins