Electrophoresis Exam – Questions With Solutions
Electrophoresis basic principle Right Ans - charged particles or molecules
when placed in an electrical field will migrate towards the opposite charge
Migration depends on Right Ans - charge, size and molecular weight, shape,
strength of electrical field, type of support media, pH and time
What is the largest effect on migration Right Ans - charge. Protein take on
charge depending on what soln. pH is = amphoteric.
Size and molecular weight Right Ans - smaller molecule migrate faster and
larger molecule migrate slower
shape Right Ans - protein denatures and will slow migration. round
(newer) go faster
strength of electrical feild Right Ans - increase voltage will increase
migration up to a point. If increase voltage too much then heat will denature
proteins and evaporate fluid which will increase conc. of buffer.
type of support media Right Ans - agar based is most common
pH Right Ans - Buffer must be the right pH to create a negative protein
charge
Time Right Ans - limit time because at some point all proteins will migrate
to the end.
Charge effect on migration: 2+ molecule will migrate 2X as far as Right Ans
- -2 charge
How many fractions for serum protein Right Ans - 5
We make protein negative (anion) so they migrate toward the Right Ans -
anode (positive)
, Bridge Right Ans - where the serum goes on the electrophoresis. Put
sample in strips with a pipette on the media.
Buffer moves up both sides by Right Ans - osmosis. Particles in buffer move
toward cathode.
Electrophoresis instrument Right Ans - power supply, support media,
buffer, bridge
After electrophoresis Right Ans - fix, stain and denseotometer to measure
Quantitate based on Right Ans - thickness of band and tell what it is based
on where it migrates
Parameters affecting electrophoresis Right Ans - physical properties of
proteins, buffer, electrical field, electro-osmosis, wick flow, support media
Proteins are amphoteric Right Ans - + and = charge
pH < pI (isoelectrical charge) Right Ans - positive charge
pH = pI (isoelectrical charge) Right Ans - no net charge. Isoelectric point
pH > pI (isoelectrical charge) Right Ans - negative charge
The further away pH is from pI Right Ans - the greater the net negative (or
positive charge)
Albumin pI Right Ans - 4.9
Gammaglobulins pI Right Ans - 7.4
In blood what charge do albumin and gammaglobulin have based on pI
Right Ans - blood pH = 7.35-7.45 --Albumin pH>pI therefore Albumin is
negative---Gammglobulin could be neutral, pos or neg. depending on exact pH
If we have a buffer with a pH of 8.6 what is charge of albumin and
gammaglobulin based on pI Right Ans - Albumin and gammaglobulin pH>
pI so both are negative. Albumin there is a greater difference so it will be more
Electrophoresis basic principle Right Ans - charged particles or molecules
when placed in an electrical field will migrate towards the opposite charge
Migration depends on Right Ans - charge, size and molecular weight, shape,
strength of electrical field, type of support media, pH and time
What is the largest effect on migration Right Ans - charge. Protein take on
charge depending on what soln. pH is = amphoteric.
Size and molecular weight Right Ans - smaller molecule migrate faster and
larger molecule migrate slower
shape Right Ans - protein denatures and will slow migration. round
(newer) go faster
strength of electrical feild Right Ans - increase voltage will increase
migration up to a point. If increase voltage too much then heat will denature
proteins and evaporate fluid which will increase conc. of buffer.
type of support media Right Ans - agar based is most common
pH Right Ans - Buffer must be the right pH to create a negative protein
charge
Time Right Ans - limit time because at some point all proteins will migrate
to the end.
Charge effect on migration: 2+ molecule will migrate 2X as far as Right Ans
- -2 charge
How many fractions for serum protein Right Ans - 5
We make protein negative (anion) so they migrate toward the Right Ans -
anode (positive)
, Bridge Right Ans - where the serum goes on the electrophoresis. Put
sample in strips with a pipette on the media.
Buffer moves up both sides by Right Ans - osmosis. Particles in buffer move
toward cathode.
Electrophoresis instrument Right Ans - power supply, support media,
buffer, bridge
After electrophoresis Right Ans - fix, stain and denseotometer to measure
Quantitate based on Right Ans - thickness of band and tell what it is based
on where it migrates
Parameters affecting electrophoresis Right Ans - physical properties of
proteins, buffer, electrical field, electro-osmosis, wick flow, support media
Proteins are amphoteric Right Ans - + and = charge
pH < pI (isoelectrical charge) Right Ans - positive charge
pH = pI (isoelectrical charge) Right Ans - no net charge. Isoelectric point
pH > pI (isoelectrical charge) Right Ans - negative charge
The further away pH is from pI Right Ans - the greater the net negative (or
positive charge)
Albumin pI Right Ans - 4.9
Gammaglobulins pI Right Ans - 7.4
In blood what charge do albumin and gammaglobulin have based on pI
Right Ans - blood pH = 7.35-7.45 --Albumin pH>pI therefore Albumin is
negative---Gammglobulin could be neutral, pos or neg. depending on exact pH
If we have a buffer with a pH of 8.6 what is charge of albumin and
gammaglobulin based on pI Right Ans - Albumin and gammaglobulin pH>
pI so both are negative. Albumin there is a greater difference so it will be more
