Transcript Electrophoresis HCC 2013 BMS2 intro

Electrophoresis
• Defined as the migration of charged particles
through a solution under the influence of an
electric field.
• Many important biological molecules possess
ionisable groups
– e.g. amino acids, peptides, proteins,
nucleotides, nucleic acids
• So, at a given pH they exist in solution as
electrically charged species either as cations (+)
or anions (-).
• If an electric field is applied charged particles
will either migrate to the cathode or anode
depending upon their charge.
Electrophoresis
Equipment for electrophoresis is a power pack and an
electrophoresis unit (gel tank) – either vertical or
horizontal.
Images from Anachem Ltd
Methodology of SDS-PAGE gels
Polyacrylamide Gels (i.e. SDS-PAGE)
* Stacking gel (4.5%)
– Stacks all the polypeptides into a narrow band
– Allows all the polypeptides to enter the separating gel at the
same time
* Separating gel (10-12%)
– Separates the various polypeptides based on their molecular weight
– The smaller the polypeptides the faster it will migrate
• The smaller the polypeptides size, the higher acrylamide concentration
needed to properly separate.
* Otherwise, the small proteins would just race through the gel matrix with
no quantitative results for classifying polypeptides.
* The best concentration for particular size ranges has thankfully been
determined by previous scientists.
Gels for Separating Proteins
Using a stacking gel
low percent
acrylamide (4%)
Resolution of good bands in resolving gel relies on all the sample entering the gel at
the same time
Molecular Weight Standards
1. Mixture of polypeptides of known molecular
weights
2. Helps to determine the molecular weight of
an unknown polypeptide
3. Does not tell you what proteins or
polypeptides are in your sample
(a) Because two proteins have the same
molecular weight does not mean they are the
same protein
(b) May be hundreds of different proteins with
the same molecular weight
Sample preparation for SDS-PAGE
Protein samples are suspended in a buffer solution
containing SDS, -mercaptoethanol, glycerol and a
tracking dye
(a) Must have excess SDS (at least 3:1 ratio)
(b) Must have excess reducing agent
Mixture is heated in a boiling water bath for a
few minutes to denature the proteins
Even when you take all precautions you must still be careful
when interpreting your results
SDS-Page
Loading the gel
Connecting to the power supply