Week 11

Question 1

What is electrophoresis

Answer 1

-separation based on charge
-proteins will have a charge because of ionization of functional groups
-NH2, COOH, R group
-current is applied
-charged particles migrate toward opposite of charged molecule
-mobility of particles is determined by the environment
-proteins have different velocities

Question 2

What are the components of PE

Answer 2

-power supply and chamber with 2 platinum electrodes
-basic buffer at pH 8.6, uses wicks to maintain contact with hydrated support medium agarose gel or cellulose acetate
-reference sample with normal and abnormal
-pt sample
-pe chamber
-detection and quantitation - fixing, drying and staining
-chamber lid prevents solvent evaporation due to heat
-Anions go to positively charged anode and cation go to negatively charged cathode

Question 3

What is iontophoresis

Answer 3

migration of small ions

Question 4

what is zone electrophoresis

Answer 4

-charged macromolecules migrate in a porous support medium like cellulose acetate, agarose gel film and polyacrylamide gel
-macromolecules can be proteins in serum, CSF and urine

Question 5

What is an electrophoretogram

Answer 5

-generated by zone electrophoresis
-creates zones or fractions of macromolecules
-physically separated from each other
-can be homogeneous (no separation step) or heterogeneous (needs a separation step)

Question 6

What is an ampholyte

Answer 6

-has acidic and basic group
-used to establish stable pH gradient as they can act as an acid or base depending on the pH

Question 7

What are the factors that affect mobility

Answer 7

-Net charge on the protein
-Size & shape of the molecule
-Ohm’s Law & power supply
-Heat production (heat produced when current flows through a medium that has resistance)
-Support media
-Time
-Ionic strength of buffer
-Electroendosmosis

Question 8

how does the “net charge on a protein “ affect mobility

Answer 8

-pI = isoelectric point (IEP)
-if pH < IEP the protein accepts H and has a overal positive charge because cations migrate to cathode (-)

pH = IEP ; net zero charge and no migration

pH> IEP ; proteins will donate H, have an overall negative charge because anions migrate to the anode (+)

the pH of the buffer around the proteins controls/alters protein charge and influences its motility

The greater the net charge of a protein, the more it is attracted to the oppositely charged electrode and the faster it moves

Question 9

how does the “Size and Shape of Molecule “ affect mobility

Answer 9

-protein motility is counteracted by frictional resistance
- based on the ionic radius of the particle
-smallest molecules move the farthest

Albumin -smallest and fastest
Globulins- large asymmetric globular and moves slower

Question 10

how does the “Ohm’s Law & power supply “ affect mobility

Answer 10

Electrophoresis is governed by Ohm’s Law: V = I R
V= electromotive force (in volts)
I = current (in amperes)
R = resistance (in Ohms)

Gel is the resistor
power supply determines current (I) and voltage (V)

Question 11

how does the “Power Supply” affect mobility

Answer 11

-keep rate of migration constant
-distance of migration (mobility) is directly related to the voltage and time (length of run)

low voltage = low mobility and vice versa
-a high voltage reduces the time to produce zone separation
-if current is constant, voltage will increase as resistance goes up
-high voltage leads to heat production because the current is moving through resistance

Question 12

how does the “Heat Production” affect mobility

Answer 12

-high voltage results in increase of thermal agitation of the dissolved solute (proteins) causing a decrease in resistance to migration and increase in current
-increase of evaporation of water from buffer which increases ion concentration of the buffer . This increases migration rate and may cause solutes to distort because of diffusion of seperated zones

-constant current keeps heat and solute diffusion low, as electrophoresis progresses a decrease in resistance also decreases the voltage

Question 13

how does the “Support Media” affect mobility

Answer 13

-support media should not bind the molecules being separated - has to be inert

Question 14

What is agarose gel like as a support media

Answer 14

-inert
-isolated from agar
-most widely used in lab
-neutral
-doesnt bind protein, therefore doesnt affect migration
-electroendosmosis reduced
-needs small amounts of sample
-dried gel can be stored indefinitely

Question 15

What is polyacrylamide gel like as a support media

Answer 15

-separates based on charge, size (due to gel pores) and weight
-layers of gel with different pore sizes
-inert

Question 16

What is Cellulose acetate
like as a support media

Answer 16

-inert
-dry brittle film with 80% air
-when soaked in buffer, air is filled with electrolyte and film becomes pliable
-can be stored for a long time
-transparent after staining

Question 17

how does the “time” affect mobility

Answer 17

-length of run in important
-the longer a run the better results are produced (good resolution)
-however longer runs can also cause increased diffusion of zones due to heat production

use minimal time to produce best seperation

Question 18

how does the “Electrophoresis Buffers” affect mobility

Answer 18

-buffers have ions to help keep pH constant and carry applied current
-determine electrical charge on molecule, magnitude of charge and direction of migration

-barbital buffers (proteins), EDTA buffers (nucleic acids)

Question 19

how does the “Ionic Strength of Buffers” affect mobility

Answer 19

-refers to charge and concentration of ions
-determines thickness of ion cloud around a molecule
-determines rate of migration
-determines sharpness or resolution of electrophortic zones

-decrease ionic strength gives increased mobility but poor buffering capability

-increase ionic strength gives decreased/slow mobility, sharper zones and more heat

Question 20

how does the “Effect of Increased Ionic Strength” affect mobility

Answer 20

-greater the ionic strength the greater the resistance to movement (decreased migration)
-cleaner fractions increased resolution

-increased heat production can denature heat labile proteins

Question 21

how does the “Electroendosmosis” affect mobility

Answer 21

• overall solvent movement
  -movement of buffer ions and buffer solvent relative to fixed support medium

Question 22

why would you order SPE

Answer 22

-Unexplained weakness, fatigue, anemia, renal insufficiency, increased ESR
-Bence Jones proteinuria, heavy proteinuria in >40 year old patients
-Hypercalcemia
-Hyper gammaglobulinemia, -immunoglobulin deficiency
-Recurrent infections

Question 23

What is SPE

Answer 23

IEP of albumin is 4.8
IEP of gamma globulin is 7.4

-use barbital buffer of pH 8.6 because at this pH all serum proteins are negatively charged and migrate toward the anode

-this buffer pH is farthest from IEP of albumin so it has the largest charge and moves the fastest

-gamma globulins have smallest net charge and move the smallest amount

-reference sample and PT samples are applied to the cathode end of gel

Question 24

how to fix and stain after electrophoresis

Answer 24

fix - immobilize the bands,
dilute acids acetic acid, trochloroacetic acid and acid alcohol

stain using
-Amido Black, Ponceau S, Coomassie Brilliant Blue
-lipoproteins (Oil Red O, Sudan Black B), or CSF proteins (silver nitrate).
-type of stain varies with what is being separated

Chemiluminescence for igE using Anti IgE labelled with enzyme _dioxetant P

Question 25

Quantitation in PE

Answer 25

-use a densitometer to quantitate fractions -% of total protein or absolute concentration (g/L) -measures reflectance or transmittance -the denser the stain on the band the more light it absorbs signal for each band is seen as peak, area under the peak are calculated and % is converted into concentration

Question 26

Densitometric scan: Serum Proteins what are the peaks seen as

Answer 26

5 peak = 5 stained zones -usually only 5 bands are seen on healthy people on an electrophoretogram. Proteins in bands can be quantitated separately by immunoassay -in diseased states bands can increase or decrease -peaks are homogenous with one protein or hetero with several -homogenous proteins like albumin give narrow band -width of fraction depends on number of proteins

Question 27

What is albumin

Answer 27

-most abundant serum protein -elevated only when dehydrated -decreases are of clinical interest - chronic liver disease, nephrotic syndrome, extensive burns, malabsorption can be non specific due to inflammatory reactions

Question 28

What is alpha 1 globulin

Answer 28

-alpha-1-antitrypsin, alpha-1-acid glycoprotein, and alpha-1-lipoprotein -increases indicate active tissue damage like inflammation, malignancy and post op conditions

Question 29

What is alpha 2 globulin

Answer 29

-consists of 3 bands alpha-2 macroglobulin (most anodal), haptoglobin, and beta-lipoprotein (most cathodal and can also run in the beta zone) -a raised fraction with a reduction of others can be nephrotic syndrome , protein losing issues -haptoglobin is variable genetically and can migrate in the same broad band as beta 2 macroglobulin instead of alone

Question 30

Nephrotic syndrome

Answer 30

decrease in albumin decrease in gamma globulins increase in alpha 2 globulins

Question 31

What is beta globulin

Answer 31

made up of transferrin beta lipoprotein complement C3 (heat labile and decreases if stored at room temp) -visible changes here are uncommon

Question 32

what gamma globulins

Answer 32

consists of IgA, IgM, IgG raised gamma globulins in Chronic infections cirrhosis of liver autoimmune disorder decreased in Nephrotic syndrome (increased loss) Malnutrition or immun0- deficiency (decreased synthesis) Secondary suppression of synthesis

Question 33

What will you have if you have an increase in a single globulin out of the three

Answer 33

Multiple myeloma Waldenstrom’s macroglobulinemia Bench-Jones proteinemia

Question 34

What will be seen on monoclonal gammopathies

Answer 34

-Disorder of Ig synthesis due to b cell clone syntesis -increase in plasma cells with single spike in M protein in beta gamma region -if there is M protein there is a decrease in normal Ig -if there is high M protein and decreased levels of Ig and can be associated with a malignant clinical course -biclonal with increase in Beta and gamma and alb seen as increase in peak at gamma region because of multiple myeloma

Question 35

what is seen in cirrhosis of liver

Answer 35

decrease of albumin increase in gamma bridging between beta and gamma

Question 36

what will you see in polyclonal gammopathy

Answer 36

=hypergammaglobulinemia -increase of all gamma globulins -decreased albumin due to chronic infection liver disease burn autoimmune disease

Question 37

what will you see in liver disease

Answer 37

decreased albumin increased gamma globulins (polyclonal) beta gamma bridging

Question 38

what is hypogammaglobulinemia

Answer 38

decreased or absent gamma globulin due to congitenal disorder (IgA or IgG def) and immunosuppressive therapy

Question 39

what will you see in viral hepatitis

Answer 39

decrease in albumin increase in gamma globulins

Question 40

What will you see in chronic inflammation

Answer 40

increase in APRs - AAT, AAC, HAP, CER, C3/4 CRP -non specific and general reaction to inflammation -each APR rises at different rates -seen with decrease in albumin synthesis so your TP will change

Question 41

how can you apply electrophoresis spinal fluid proteins

Answer 41

-agarose gel electrophoresis -shows albumin as major band as it makes up most of the CSF protein -there will be a extra pre albumin fraction -pre alb, alb, antityrp, transferrin, csf specific transferrin -on abnormal patient you will see OLIG bands

Question 42

What are OLIG banding in spinal fluid proteins

Answer 42

-seen in gamma fraction in pt with MS -can be seen very early and will be in the bottom of gel toward you

Question 43

how can you apply electrophoresis isoenzymes

Answer 43

, LD, ALP, CK -different types of LD banding indicates which organ is damaged LD 1 - LD 5 increase in1,2, 3, 5 for MI Increase in 1, 2, and 3 for pernicious or hemolytic anemia increase in 4 and 5 = active liver disease increase in all = malignant tumor shock Alp comes from the liver, bone and intestine there is one band where liver and bone ALP overlaps

Question 44

how can you apply electrophoresis isoenzymes CK

Answer 44

found in cardiac, brain and skeletal muscles -if there is heart and brain damage you will see 3 distinct electrophoretic bands CKMM increase Excessive Muscular activity CKMM and ckmb increase - MI CKMM and a tiny CKMB = duchennes muscular dystrophy

Question 45

how can you apply electrophoresis Lipoproteins

Answer 45

-lipids are transported in plasma -chylomicrons transport dietary lipids which will not be seen in fasting patient made up of : -pre beta transports endogenous lipids -B transports cholesterol -Alpha is made up of protein bound phospholipids lipoproteins can be separated into 5 types 1-5

Question 46

What to watch for in electrophoresis

Answer 46

stain solution -replace if they are leaching or if there is staining -stains should be stored in tight containers to avoid evaporation Unequal migration rates -dirty electrodes which support drying out use small samples to avoid overloading with albumin because it is 10x more concentrated than alpha 1 and we need to be able to detect this as well

Question 47

What to watch for in electrophoresis band irregularities

Answer 47

-distortion because of bent applications, bubbles, a wet or dry support -artefacts, hemolysed samples, fibrinogen, split albumin or a widened albumin zone can cause unusual bands -denatured proteins can give atypical band make sure you use controls so you dont mistake these for reportable band

Question 48

What to watch for in electrophoresis buffers

Answer 48

-buffers can be good culture media so keep them refridgerated because cold buffers improve resolution and minimized evaporation -throw out small buffer volumes because they can cause pH changes due to electrolysis of water during electrolysis -large volumes will not be as affected

Question 49

What will hemolysed samples be seen as

Answer 49

-increase in b2 area where the hemoglobin migrates or a band between a2 and b2 from the Hb-HAP complex

Question 50

What will fibrinogen in samples be seen as

Answer 50

show near application point which is why we use serum samples to avoid this extra band

Question 51

why will you see a spilt albumin peak or a wide peak

Answer 51

wide - drug that is albumin bound split - benign condition called bisalbuminemia

Question 52

What will you have to do before putting the sample in the gel

Answer 52

blot twice ; once after removing from package and next to remove excess sample -make sure you leave time for samples to diffuse into gel before blotting let dry before you prep for staining

Question 53

What will you see in Hb electrophoresis

Answer 53

hbA with small HbF and HbA2 the HbA will migrate to beta region

Question 54

What is HBA1C

Answer 54

slow and irreversible assesses degree of glycemic control in a diabetic patient -when glucose reacts with amino group in Hb it implies high blood glucose and poor control A1c depends on RBC lifespan and glucose levels in blood -youll be able to see glycemic levels over the past 6-8 weeks

Question 55

how will heat affect gets

Answer 55

evaporates solvent from the ends of support medium -drying will cause "wick up" on both ends of support to replace moisture loss