electrophoresis

what is electrohporesis

technique used for separation and purification of ions, proteins, and other molcules

what is electrophoresis most commonly used for in the clinical laboratory

to separate the serum proteins, isoenzymes, lipoproteins, and hemoglboins in various body fluids

what does electrophoresis involves in the movement of

charged particles when an external electrical current is produced in liquid evironment

how is the electric field applied to solution

oppositely charged electrodes place in the solution.

cations moves towards

negatively charged electrode

anions move towards

positively charged electrodes

what are organic molecules?

amphoteric

charge in solution and magnitiude of charge can be altered by varying the pH

what is amphoteric

can be either positive or negative

what factors affect migration?

migration of charge

size of molecule

propertiesof support medium

strength of electric field

endosmosis

ionic strength of the buffer

temperature

migration factor:

migrationof charge

greater net charge = faster molecule moves

less net charge = slower molecule moves

migration factor:

size of molecule

large = slower

small = faster

migration factor:

properties of support medium

pores, filter the separtation not only by charge but by size

increase viscosity = slower migration

decrease viscosity = faster migration

migration factor:

strength of electric field

greater voltage applied = faster migration

migration factor:

enodomosis (limiting factor)

support medium will absorb some ion in buffer causing ion cloud

can slow stop or push migration backwards

migration factor:

ionic strength of buffer

lower buffer = faster migration

increase strength = slow migration

mirgraton factor:

temperature

increase temperature = faster migration

but too high of tempature can denature the protein

what are the components of electrophoresis

sample applicator

support medium

buffer system

electrophoresis chamber

electrodes

timer

power supply

stains

what does the buffer system do in components of electrophoresis

set the pH for migration to occur in

what are the functions of buffers?

fix and maintain a constant pH that determines the net charge on the molcules and controls the direction of migration

conduct current

what is the ideal pH for protein?

alkaline pH = protein takes a negative charge and migrates towards anode (positive charge)

8.6

what are the important consideration for buffers?

1. buffer must not reactwith sample

2. pH chosen should charge but not denature molecule

3. ionic strength of buffer must be considered

what does a slower separation result in?

result in higher resolution more distinct band separation

what are the 2 most commonly used buffers?

barbital buffer

tris-boric acid-EDTA buffers

what types of support media does electrophoresis use?

membrane or gel

when does endosmosis occur

when support medium becomes negatively charged b/c of the adsorption of hydroxyl ions from the buffer.

how does the ion cloud happen?

the negatively charged surface attracts positive ions from the buffer. ion cloud forms

depending on the strength of the charge on the molecule and the magnitude of the endomosis, the proteins can be slowed remain immobile or be pushed towards the cathode

forms friction and inhibitrs normal migration

what is wick flow?

the upward movement of buffer through both immersed ends of the gel membrane to replace lost moisture from heat generation.

what does the flow of wick flow add to?

adds distance to molcule moving towards the center of the membrane and slows molecules moving aways from the center.

what is the net result of wick flow?

results in a reduced separation and lead to compression of the final band patterns

not clean band. all glob together in the middle

what are the types of support medias?

agarose gel

cellulose acetate

polyacrylamide gel

starch gel

what is the most widely used support media?

agarose gel

what is agarose gel made up of?

composed of agrose and agaropectin

what is agarose gel free of and so what does not occur?

ionizable groups

so little endosmosis occurs

what does agarose gel give better results than cellulose acetate?

sharper results

what sample size will agarose gel accept?

will accept larger sample size

what is an advantage of agarose gel?

optically clear, used for densitometry immediately (measurement of band)

what is densitometry?

use to measure the amount of compound in each band

what needs to happen to media if it is cloudy or turbid?

needs a clearing step with a solvent before measuring bands

what is cellulose acetate composed of?

cellulose and acetic anhydride

how does cellulose acetate start as?

brittle, film of interlocking cellulose acetate fibers

what must cellulose acetate be done first before starting electrophoresis?

presoak in buffer to make membrane pliable

how are the bands in cellulose acetate?

protein absorption is minimal, and sharp, well-defined bands are obtained

what is the strength and pore size of cellulose acetate?

tends to be strong, and have uniform pore size

what is the color of cellulose acetate media?

opaque, so solvent must be used to clear media for densitometry, when solvent is used, it partially dissolves the cellulose acetate fibers leaving the stained fraction on the transparent background

what is polyacrylamide gel limited to?

limited to separation of alkaline phosphatase isoenzymes

how is polyacrylamide gel formed?

by the reaction of “activated” acrylamide molecules that produce long polymer chains.

what does polyacrylamide gel chain require?

requires that the chains be cross-link with one another

what happens if the concentration of acrylamide is adjusted?

the pore size can be varied which determines the extent of cross-linkage

what happen when the pore size is large in polyacrylamide gel?

almost all serum protein will migrate unhindered, and separation will be according to charge

what happens when pore size is smaller in polyacrylamide gel?

moleculare sieving becomes a factor as larger molecules are slowed down and separation is not only by charge but by size

what is polyacrylamide gel considered as now?

carcinogen

what is starch gel composed of?

composed of granules containing amylose and amylopectin

when a concentrated suspension of the gransules is heated a viscous solution is formed and becomes a gel upon cooling

starch gel separates molecules based on what?

based on size and charge

why is starch gel not consistant?

due to the wide range of pore sizes, consistency is difficult to achieve

harder to obrain reproducible results

what stains are used in electrophoresis?

ponceau S

Coomassie Brilliant Blue

Oil Red O

Schiff’s reagent

SyBr

coupled rxn

what is the most common stain for electrophoresis?

Ponceau S

what medium is Ponceau S used for

cellulose acetate x protein separation

bright red

what media is coomassie brilliant blue used for?

polyacrylamide gel x alkaline phosphatase isoenzymes

bright blue

what is Oil Red O

is a fat stain, fatty acid will absorb it

red color

phospholipids, lipoproteins will absorb stain

what is Schiff’s reagent for?

lipoprotein and glycoproteins

what is SyBr

used for separation of DNA and RNA

common colors are gold but also green

how are coupled reactions used in electrophoresis

it generates a product that can be detected by fluorescence or by its color

amount of fluorescence or amount of color is proportional to how much in the band

what is the most common specimen for electrophoresis?

serum and plasma - most common

following urine and CSF

what other body fluids are used in electrophoresis?

pleura, pericardium, peritoneum, synovial fluid, and tears

what may a sample be required to before electrophoresis?

pretreatment depending on concentration of molecules to be separated

possible interfering substances and support medium used and information to be collected

insouble sample need to pretreat to make soluble

what pretreatment does fluids like urine and csf need?

small qty of protein, need to concentrate protein

what pretreatment does fluids like synovial fluid?

often vry thick so needs to be thinned

what pretreatment does fluids like insoluble or aggresgating molcule

need to make soluble

source of error:

problems with sample application

over fill well causes distorted band

not enough causes incomplete migration

source of error:

buffer stability

expired contaminated causes shorten migration

refigeration to decrease bacterial growth and increase shelf stability

source of error:

support media preparation and/or contamination

making own media = increase error avoid fingerprints before it can dry

presoak, put media in slowly so no air bubbles can distort band

wet media can cause distorted band

source of error:

stain issue

leaching, coming off the protein = stain is old and needs replacement

what are the 2 negative phase reactant?

albumin and Transferrin

where do most of our antibodies lie in?

gamma region (largest %)

where are IgA, IgM, and IgG located on?

small % on the right/far side of beta region

normal

increase gamma spike (IgM)

multiple myeloma

monoconal spike/gamopathy

increased IgA

beta-gamma bridge

cirrous

CRP elevated located between beta and gamma region no separation

alpha 1 lower, alpha 2 higher, beta and gamma lower

nephrotic syndrome

alpha 2 have most positve acute phase reactents

elevation in alpha 1 and 2

inflammation

acute phase reaction or immediate phase reaction

recent infection because gamma not elevated (immune not involved yet)

immune reaction

on the right

reduced gamma region

immunocompromised, unable to produce antibodies

what are some examples of immunocompromised

treated for cancer, chemotherapy or drug that wipes out immune system

transplants, anti-rejection med

steroids lowers immune system

AIDS

what is the immunocompromised pattern also called?

hypogammaglobulinamia

acute phase reactant

immedate reaction

immune response

rounded gamma

immunocompromised,

no gamma region

cirrous

beta gamma bridge

multiple myeloma

more spikey

nephrotic syndrome

decrease albumin and gamma

increase alpha 2

Protein-losing enteropathy - PLE

losing protein in feces

similar to nephrotic

how can you tell the difference between PLE and nephrotic syndrome on electrophoresis

nephrotic - inflammatory condition, higher alpha 2, lower gamma