electrophoresis Flashcards
What are the two kinds of electrophoresis?
- Zone electrophoresis (majority)
- Moving Boundary Electrophoresis
What is Zone electrophoresis?
Moving proteins through a solid medium/matrix.
What are the types of Zone electrophoresis?
- Paper electrophoresis (cellulose acetate)
- Gel electrophoresis (agarose or polyacrylamide gel)
What is Moving Boundary Electrophoresis?
Moving proteins through an aqueous type of medium.
What are the types of Moving Boundary Electrophoresis?
- Capillary electrophoresis
- Immuno electrophoresis
What are the components of electrophoresis?
- Driving force - power supply
- Support medium - agarose gel, etc.
- Buffer
- Sample
- Detection System
What role does the buffer play in electrophoresis?
Serves as an important component; connects the sample and electrical force, dictates pH.
What happens to proteins at different pH levels?
pH > pI: Negative charge
pH < pI: Positive charge
What is the goal of electrophoresis?
To ensure all proteins have the same charge.
What is the charge state of most proteins?
Most are isoelectric: neutral and will not be moving.
Why do negatively charged proteins absorb less water?
Negatively charged proteins absorb less water and buffer than positively charged proteins.
Which direction do serum proteins migrate?
Serum proteins migrate toward the anode, starting at the cathode.
What factors affect the movement of proteins?
- Weight of protein
- Velocity (depends on charge and strength of electrical field)
- Medium used
- Temperature
What is the maximum temperature for maintaining proteins during electrophoresis?
Heat cannot be above 55 degrees; will denature proteins.
What is the importance of ionic strength in electrophoresis?
Ionic strength directs migration; too low enhances movement, too high makes it sluggish.
What is the normal ionic strength range for electrophoresis?
Normal strength = 0.01 - 0.1
What materials are used for electrophoresis?
Proteins easily migrate in cellulose acetate and/or agarose gel.
maintain even temp
do not absorb water or buffer from system
prevent endo electrical osmosis
What is the advantage of polyacrylamide gel?
More separation of proteins; separates based on molecular weight.
called poly because of diff layers of agarose gel
What is a common specimen used in electrophoresis?
Serum, diluted in buffer.
can also use urine /CSF
-no dilution; concentrate then
What is the typical volume of specimen used in electrophoresis?
Use 2-5 mL of specimen.
What should be done before running electrophoresis?
Plug in the power supply before putting proteins in (like warm up the oven.)
What are common detection methods in electrophoresis?
Stains such as ponceau (cellulose acetate) and coomassie blue (any agarose gel).
What is bromocresol purple used for?
Used in place of coomassie blue at pH 5.2 (proteins positively charged).
What is bromocresol green/yellow dye used for?
Used for detection of albumin.
what is endo electrical osmosis
backward migration of proteins from starting ??
proteins migrate better if they have
negative charge
order of bands from cathode to anode
gamma
B1 and B2
A2
A1
Albumin
normal total serum protein value
6-8 gm/ dL
normal AG ratio
0.8-1.8
what is the highest peak in electrophoresis
albumin
what is 1st protein lost in kidney disfunction
albumin
2 primary roles of albumin
- helps maintain osmotic pressure
- transport protein
reasons for decreases in albumin
liver dysfunction- albumin made here
kidney disfunction
inflammatory/ infectious processes - making more APR
reason for increase in albumin
dehydration
reason for bisalbuminemia (teak peaks)
hereditary, greek culture, drugs
non pathologic event
main protein seen in alpha 1 region
alpha 1 anti-trypsin
purpose of alpha 1 anti-trypsin
Protein that will help with lung function; inhibits some protease that can be seen in certain diseases
can be APR
decreases in alpha 1 anti-trypsin are
asthma, etc
problems with lungs
other protein in alpha 1 region
alpha 1 fetoprotein
-low levels after birth??
-tumor marker
what is alpha 1 region home to
APR- increases during infections
alpha 2 region main protein
haptoglobin - first thing measured in hemolytic event
another protein found in alpha 2
ceruloplasmin- binds copper
increased levels of copper shown in
wilson’s disease - absorption not regulated
decreased ceruloplasm
will not get lost in kidney: too large to pass
alpha 2 macro globulin
-protective to fetus with mother’s immune response
beta region protein
transferrin - binds 2 atoms of iron
iron overload and muscle damage; hemochromatosis = decrease
transferrin (all iron bound)
inflammation and IDA = increase
transferrin
another protein in beta region
CRP- c reactive protein
APR
CRP only increases in
acute inflammatory (now)
gamma globulins made in
gamm region with plasma cells
normal or decreases albumin
-increase A1 and A2; APR
acute inflammatory response
- Normal or decreased albumin
- Increase A1 or A2 region
- Increase in gamma region
- Sometimes beta
chronic inflammatory response
- Plateau in the gamma region
- Over production of antibodies??; auto immune diseases
- Not a cancerous event; it’s a normal event
May be transient
polyclonal gammopathy
- M spike or church spike/ peak
- Can be higher than albumin
- Be toward the cathode end ; gamma
- Cancerous event
- Overproduce 1 type of immunoglobulin
○ Non functioning antibodies from oncogene
○ Ex. Multiple myeloma- See presence of Bence jones proteins: free light chains
monoclonal gammopathy
hypogammaglobulinemia
not making antibodies
B cell defect
give them IV gamma globulins
characteristic of alcohol cirrhosis of the liver
beta- gamma bridge
regions blur together
GGT and osmol gap increased
SPE
serum protein electrophoresis
SPE is based on 2 principles
1) electrophoresis
2) precipitation
steps serum protein electrophoresis
1) ID presence of monoclonal gammopathies or polyclonal gammopathies or normal
look for increase IgG, IgA, IgM, kappa, and lambada
specimen used for SPE
serum and urine
done with agarose gel
how to perform SPE?
specimen placed cathode 2-5
allow electrophoresis 15-30 min
once migrate, add anti-serum to see if precipitation band; incubate
detection: add stain to read
polyclonal apperance on test
smudging
monoclonal appearance on test
distinct line
what do you look for in urine electrophoresis?
bence jones proteins
-over production of free light chains
in liver disease graph
everything decreases besides gamma because not produced there
kidney disease graph
alpha 2 increases; macroglobulin
-can’t pass kidney
inflammatory graph
APR increases
albumin normal or slightly decreased
spinal fluid normal protein
15-45
measure of nutrition in CSF electrophoresis
prealbumin
how many bands in normal CSF
7
-beta regions more distinct
hemorrhaging in CSF graph
increase in B2 because increase in fibrinogen
HGB alkaline used as a
screen
if something abnormal in alkaline use
acid
