Electrophoresis Flashcards
What is electrophoresis
the movement of charge particles by an external electrical field
What is the rate of migration during electrophoresis dependant on
the properties of the support media, electric field strength and temperature
What does electrophoresis do
separate and analyze proteins and nucleic acids
What is agarose
a support medium made of purified agar
How is the pore size of agarose gel controlled
by concentration
How do proteins separate in electrophoresis
they separate into bands based on their mass to charge ratio
How does DNA usually migrate through an agarose gel
based on size since they all have the same charge
Why is agarose preferred to other methods
it is easy to handle, has no charge, and contributes minimally to electroendosmosis
What is electroendosmosis
charged components of the support media attract counterions from the buffer which move towards the oppositely charged electrode taking the solvent with them, this forces the net movement of solvent in one direction which can slow down or reverse analyte migaration
What does the pH of a buffer do
determines the net charge of the analytes during electrophoresis
How does the rate of migration in a system change as conductivity of the buffer increases
it decreases
How do we keep the current constant
we decrease the voltage which decreases the applied electrical field
How do we maintain constant voltage
increase the current as the ionic strength of the buffer increases
What is the risk of a high current
overheating and convection currents
What effect does pH have on electrophoresis
determines the charge of analyte and therefore its mobility
What effect does ionic strength have on electrophoresis
alters voltage, increased ionic strength usually reduces migration rate, increased ionic strength usually increases heating
What effect does current have on electrophoresis
too much current results in excessive heat production
What effect does voltage have on electrophoresis
migration rate is proportional to voltage
What effect does temperature have on electrophoresis
temperature gradients cause curved bands, excess heat can denature proteins, lower temperatures decrease migration rates
What effect does time have on electrophoresis
resolution of bands increases with time
What effect does support media have on electrophoresis
electroendosmosis and pore size effect migration rates
How does DNA migrate in electrophoresis
from the cathode to the anode
What is an ampholyte
something that has both a positive and negative charge
What effect does the isoelectric point have on a protein
it causes it to have no net charge
How does pH above the isoelectric point do to a protein
causes it to be negatively charged
How does pH below the isoelectric point do to a protein
causes it to be positively charged
What are the conditions for serum protein electrophoresis
alkaline pH above the isoelectric point of all proteins allowing them all to be negatively charged
What occurs after serum protein electrophoresis
proteins are fixed in the agarose and stained
What are common dyes for SPE
coomassie brilliant blue, amido black and ponceau S
What causes unequal migration across the well
dirty electrodes, uneven wetting of the gel
What causes distorted protein zones
bent applicator, bubble introduced during sample application, too much sample applied
What causes usual bands
hemolyzed sample, plasma sample, medication
How is capillary electrophoresis carried out
in narrow, less than 500nm fused silica capillary tubes up to twenty meters in length
How are capillaries reinforced
exterior coating of polyimide
What kind of sample injection is used for capillary electrophoresis
hydrodynamic injection
How does hydrodynamic injection work
one end of the capillary is placed in the sample and differential pressure is applied
How do proteins move in capillary electrophoresis
freely in alkaline solution without supporting medium
What causes an electroendosmosis force in capillary electrophoresis
the negatively charged silanol groups of the fused silica capillary which attract counterions from the buffer
What direction do proteins migrate in capillary electrophoresis
towards the cathode
How are proteins separated in capillary electrophoresis
based on their electrophoretic mobility and endosmosis
What is an important step for DNA capillary electrophoresis
DNA must be labelled with fluorescent labeled nucleotides before separation
How does DNA capillary electrophoresis work
DNA fragments move through a flowable polymer of polyacrylamide at an alkaline pH, the interior of the capillary is coated to eliminate electroendosmosis
How is DNA separated in capillary electrophoresis
the flowable polymer acts as a molecular sieve to separate the nucleic acids based on length
How does DNA move through capillary electrophoresis
injected at the cathode and moves to wards the anode
