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84 Cards in this Set
- Front
- Back
Define Electrophoresis |
migration of a charged particle through an electric field |
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2 main types of Electrophoresis |
1) moving boundary 2) Zone |
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Which electrophoresis method moves through aqueous/liquid medium? |
moving boundary |
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Which electrophoresis method moves through solid medium? |
zone |
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Solid mediums used in zone electrophoresis? |
1) agarose gel 2) cellulose acetate 3) acrylamide gels 4) paper |
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Why are agarose gels and cellulose acetate the most common solid mediums in zone electrophoresis? |
1) they are easy to make 2) proteins migrate readily 3) medium don't take on charge themselves |
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5 main parts of Electrophoresis systems |
1) driving force 2) support medium 3) buffer 4) sample 5) detection system |
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Amphoteric |
proteins carry both a positive and negative charge |
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Why can't proteins be amphoteric in electrophoresis? |
they move based on charge so they have to have only one charge |
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Isoelectric Point (PI) |
number of negative charges = number of positive charges |
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How can altering pH chance PI? |
pH > PI = negative protein PI > pH = positive protein |
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Why do we want proteins to be negatively charged rather than positively charged in electrophoresis? |
positive charges absorb more moisture but we don't want proteins to absorb in electrophoresis |
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How do positively charged and negatiively charged proteins move in electrophoresis? |
positive proteins move toward the negative cathode negative proteins move toward the positive anode |
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What pH of buffers are used in electrophoresis? |
use a buffer with a pH > 8 (usually 8.6) so that pH > PI so that proteins are negatively charged and move toward the positive anode |
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How do buffers effect ions in protein electrophoresis? |
maintain ionic strength of the system to help ions carry current |
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How does ionic strength effect protein electrophoresis? |
if ionic strength is LOWERED, proteins will migrate faster and bands will be more separated if ionic strength is INCREASED, proteins will migrate faster and bands will be closer |
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Normal ionic strength in protein electrophoresis? |
.5 |
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What can happen if ionic strength is too low in protein electrophoresis? |
the temperature can increase too much and denature proteins |
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What should protein electrophoresis temperature stay below? |
50 degrees |
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How does voltage effect protein electrophoresis? |
increased voltage moves proteins faster decreased voltage moves proteins slower |
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What can happen in voltage is too high in protein electrophoresis? |
heat can increase too high which may cause proteins to denature and buffers to evaporate |
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What is endoelectroosmosis and what solid media can prevent it? |
buffer/ions push proteins in wrong direction against negative to positive movement, it can cause the gamma region to be behind the point of origin agarose gel and cellulase acetate |
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Benefits of cellulose acetate solid material |
easily stained white paper that separates proteins into 5 bands, easy to read and can be kept forever |
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Benefits of agarose gel solid material |
gel is porous so proteins can move easily, can bee stained easily and kept forever |
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Disadvantages of agarose gel solid material |
touching the gel can effect migration, may get background staining |
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How are proteins separated in polyacrylamide gel? |
based on molecular weight rather than charge, can separate proteins into 20+ fractions |
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WHat is added to proteins in polyacrylamide gel technique? |
neutralizing agent so proteins don't charge |
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What should you dilute proteins specimens in for electrophoresis? |
buffer *except urine and CSF- shouldnt have much protein |
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How much specimen is used for protein electrophoresis? |
2-5 uL, put into little slits so overfilling is prevented |
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How long does most protein electrophoresis take? |
<30 min, prevent bands from being smushed or run off |
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Main stains used for protein electrophoresis |
1) amido black 2) amido blue 3) ponceau S 4) coomassie blue |
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Main stains used for lipid electrophoresis? |
1) sudan 2) oil red O |
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Bands of serum protein electrophoresis |
1) albumin 2) alpha 1 3) alpha 2 4) Beta 5) gamma |
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What protein makes up 60% of the 6-8 g/dL total protein? |
albumin |
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Where is albumin synthesized? |
liver |
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What are the main functions of albumin? |
osmotic pressure, transportation |
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Causes of decreased albumin |
impaired liver function (synthesis) kidney disease (loss) malnutrition infectious disease |
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#1 reason for LOW anion gap |
hypoalbuminemia |
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In liver disease, all proteins except ____ will decrease? |
gamma globulin |
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In kidney disease, all proteins will decrease except___? |
alpha 2 region |
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Cause for increase in albumin |
relative to dehydration |
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Bisalbuminemia |
2 peaks in albumin region, caused by genetics or drugs: not a problem |
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Main protein in alpha 1 region |
alpha 1 antitrypsin (90%) |
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Job of alpha-1 antitrypsin |
maintains elasticity of lungs in infection, may be genetic |
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What can decreased levels of alpha 1 antitrypsin do? |
cause lungs problems in 20s-30s |
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When can alpha-1 antitrypsin levels be elevated? |
acute infections, chronic infections |
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What is the main protein in the alpha 2 region? |
haptoglobin |
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Main job of haptoglobin |
binds to free hemoglobin to save it |
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When is haptoglobin decreased? |
hemolytic event |
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What region is ceruloplasmin in? |
alpha 2 |
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What does ceruloplasmin do? |
binds copper |
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When is ceruloplasmin decreased? |
Wilson's disease (will see kaiser fletcher rings) |
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What is alpha 2 macroglobulin? |
a large acute phase reactant that can aid in growth and transport insulin |
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Why isn't the alpha 2 region decreased in kidney failure? |
alpha 2 macroglobulin is too big to be excreted |
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When is alpha 2 macroglobulin increased? |
infections, inflammatory processes |
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Main protein in the beta region? |
transferrin |
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Job of transferrin |
binds/transports 2 molecules of iron |
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When is transferrin increased? |
iron deficiency anemia |
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When is transferrin decreased? |
iron overload (hemochromatosis or hemosiderosis) |
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What region is C reactive protein in and when is it increased? |
beta, infectious process |
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When can gamma globulins be increased? |
monoclonal/polyclonal gammopathy, infectious process |
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When can the gamma region be decreased> |
kidney disease |
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What antibodies will commonly migrate to the beta region in a monoclonal gammopathy? |
IgM, IgA |
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What indicates monoclonal and polyclonal gammopathies? |
mono: M spike poly: plateau |
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What does beta-gamma bridging indicate? |
liver cirrhosis |
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What is commonly seen in plasma electrophoresis vs serum? |
fibrinogen and clotting factors, seen in beta region |
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Why must you concentrate spinal fluid for electrophoresis? |
it doesnt have as much protein (only .4 g/L) |
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How much spinal fluid is in an adult? |
90-150 mL |
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What regions can be present on a spinal fluid electrophoresis? |
pre-albumin/transerythrotin beta 2 |
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What region do you look at in spinal fluid for a demyelinating disease? |
gamma |
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Oligoclonal banding |
increase in gamma region of spinal fluid (NOT SERUM) that indicates demyelinating disease |
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If oligoclonal banding is also seen in serum, what can it be? |
toxoplasmosis, neurosyphilis |
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What can increases in spinal fluid albumin indicate? |
broken BBB |
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IgG index |
indicates demyelinating disease: IgG syntesized in spinal fluid CSF IgG/serum IgG ___________________ CSF alb/serum alb |
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Normal IgG index |
.3-.7 |
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How do you know if there is too much albumin in the spinal fluid? |
CSF albumin/serum albumin should be <.65% or <9 ????? |
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2 main principles of immunofixation/IFE |
1) electrophoresis: separate proteins via charge 2) precipitation: take soluble antigen/antibodies to make an insoluble complex |
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What happens in IFE? |
globulins become fixed to an agarose gel |
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Buffer of IFE? |
8.6 pH |
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What are the main sections in immunofixation? |
whole serum, IgG, IgA, IgM, K, l |
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WHat will mess up IFE? |
if you touch the agarose gel |
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Steps in immunofixation |
1) put specimen at bottom (right amount in slit) 2) turn on and electrophorese about 1/2 hour, become "antigens" 3) precipitate by adding "anitbodies"/antiserum to make Ab-Ag complex in troughs 4) incubate 5) stain |
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What was used before immunofixation and why isnt it used anymore? |
immunoelectrophoresis (IEP): hard to read |
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How does IEP work? |
same as IFE: in buffer 8.6 pH, migrate toward anode, electrophoresis and precipitation *but there is a pt. and a normal on each side and you have to read the thickness of the bands |