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84 Cards in this Set

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Define Electrophoresis

migration of a charged particle through an electric field

2 main types of Electrophoresis

1) moving boundary




2) Zone

Which electrophoresis method moves through aqueous/liquid medium?

moving boundary

Which electrophoresis method moves through solid medium?

zone

Solid mediums used in zone electrophoresis?

1) agarose gel


2) cellulose acetate


3) acrylamide gels


4) paper



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

5 main parts of Electrophoresis systems

1) driving force


2) support medium


3) buffer


4) sample


5) detection system

Amphoteric

proteins carry both a positive and negative charge

Why can't proteins be amphoteric in electrophoresis?

they move based on charge so they have to have only one charge

Isoelectric Point (PI)

number of negative charges = number of positive charges

How can altering pH chance PI?

pH > PI = negative protein




PI > pH = positive protein

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

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

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

How do buffers effect ions in protein electrophoresis?

maintain ionic strength of the system to help ions carry current

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

Normal ionic strength in protein electrophoresis?

.5

What can happen if ionic strength is too low in protein electrophoresis?

the temperature can increase too much and denature proteins

What should protein electrophoresis temperature stay below?

50 degrees

How does voltage effect protein electrophoresis?

increased voltage moves proteins faster




decreased voltage moves proteins slower

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

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

Benefits of cellulose acetate solid material

easily stained white paper that separates proteins into 5 bands, easy to read and can be kept forever

Benefits of agarose gel solid material

gel is porous so proteins can move easily, can bee stained easily and kept forever

Disadvantages of agarose gel solid material

touching the gel can effect migration, may get background staining

How are proteins separated in polyacrylamide gel?

based on molecular weight rather than charge, can separate proteins into 20+ fractions

WHat is added to proteins in polyacrylamide gel technique?

neutralizing agent so proteins don't charge

What should you dilute proteins specimens in for electrophoresis?

buffer




*except urine and CSF- shouldnt have much protein

How much specimen is used for protein electrophoresis?

2-5 uL, put into little slits so overfilling is prevented

How long does most protein electrophoresis take?

<30 min, prevent bands from being smushed or run off

Main stains used for protein electrophoresis

1) amido black


2) amido blue


3) ponceau S


4) coomassie blue

Main stains used for lipid electrophoresis?

1) sudan


2) oil red O

Bands of serum protein electrophoresis

1) albumin


2) alpha 1


3) alpha 2


4) Beta


5) gamma

What protein makes up 60% of the 6-8 g/dL total protein?

albumin

Where is albumin synthesized?

liver

What are the main functions of albumin?

osmotic pressure, transportation

Causes of decreased albumin

impaired liver function (synthesis)


kidney disease (loss)


malnutrition


infectious disease

#1 reason for LOW anion gap

hypoalbuminemia

In liver disease, all proteins except ____ will decrease?

gamma globulin

In kidney disease, all proteins will decrease except___?

alpha 2 region

Cause for increase in albumin

relative to dehydration

Bisalbuminemia

2 peaks in albumin region, caused by genetics or drugs: not a problem

Main protein in alpha 1 region

alpha 1 antitrypsin (90%)

Job of alpha-1 antitrypsin

maintains elasticity of lungs in infection, may be genetic

What can decreased levels of alpha 1 antitrypsin do?

cause lungs problems in 20s-30s

When can alpha-1 antitrypsin levels be elevated?

acute infections, chronic infections

What is the main protein in the alpha 2 region?

haptoglobin

Main job of haptoglobin

binds to free hemoglobin to save it

When is haptoglobin decreased?

hemolytic event

What region is ceruloplasmin in?

alpha 2

What does ceruloplasmin do?

binds copper

When is ceruloplasmin decreased?

Wilson's disease (will see kaiser fletcher rings)

What is alpha 2 macroglobulin?

a large acute phase reactant that can aid in growth and transport insulin

Why isn't the alpha 2 region decreased in kidney failure?

alpha 2 macroglobulin is too big to be excreted

When is alpha 2 macroglobulin increased?

infections, inflammatory processes

Main protein in the beta region?

transferrin

Job of transferrin

binds/transports 2 molecules of iron

When is transferrin increased?

iron deficiency anemia



When is transferrin decreased?

iron overload (hemochromatosis or hemosiderosis)

What region is C reactive protein in and when is it increased?

beta, infectious process

When can gamma globulins be increased?

monoclonal/polyclonal gammopathy, infectious process

When can the gamma region be decreased>

kidney disease

What antibodies will commonly migrate to the beta region in a monoclonal gammopathy?

IgM, IgA

What indicates monoclonal and polyclonal gammopathies?

mono: M spike




poly: plateau

What does beta-gamma bridging indicate?

liver cirrhosis

What is commonly seen in plasma electrophoresis vs serum?

fibrinogen and clotting factors, seen in beta region

Why must you concentrate spinal fluid for electrophoresis?

it doesnt have as much protein (only .4 g/L)

How much spinal fluid is in an adult?

90-150 mL

What regions can be present on a spinal fluid electrophoresis?

pre-albumin/transerythrotin




beta 2

What region do you look at in spinal fluid for a demyelinating disease?

gamma

Oligoclonal banding

increase in gamma region of spinal fluid (NOT SERUM) that indicates demyelinating disease

If oligoclonal banding is also seen in serum, what can it be?

toxoplasmosis, neurosyphilis

What can increases in spinal fluid albumin indicate?

broken BBB

IgG index

indicates demyelinating disease: IgG syntesized in spinal fluid




CSF IgG/serum IgG


___________________


CSF alb/serum alb

Normal IgG index

.3-.7

How do you know if there is too much albumin in the spinal fluid?

CSF albumin/serum albumin should be <.65% or <9




?????

2 main principles of immunofixation/IFE

1) electrophoresis: separate proteins via charge


2) precipitation: take soluble antigen/antibodies to make an insoluble complex

What happens in IFE?

globulins become fixed to an agarose gel

Buffer of IFE?

8.6 pH

What are the main sections in immunofixation?

whole serum, IgG, IgA, IgM, K, l

WHat will mess up IFE?

if you touch the agarose gel

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

What was used before immunofixation and why isnt it used anymore?

immunoelectrophoresis (IEP): hard to read

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