Week 11 Flashcards
What is electrophoresis
-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
What are the components of PE
-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
What is iontophoresis
migration of small ions
what is zone electrophoresis
-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
What is an electrophoretogram
-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)
What is an ampholyte
-has acidic and basic group
-used to establish stable pH gradient as they can act as an acid or base depending on the pH
What are the factors that affect mobility
-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
how does the “net charge on a protein “ affect mobility
-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
how does the “Size and Shape of Molecule “ affect mobility
-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
how does the “Ohm’s Law & power supply “ affect mobility
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)
how does the “Power Supply” affect mobility
-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
how does the “Heat Production” affect mobility
-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
how does the “Support Media” affect mobility
-support media should not bind the molecules being separated - has to be inert
What is agarose gel like as a support media
-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
What is polyacrylamide gel like as a support media
-separates based on charge, size (due to gel pores) and weight
-layers of gel with different pore sizes
-inert
What is Cellulose acetate
like as a support media
-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
how does the “time” affect mobility
-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
how does the “Electrophoresis Buffers” affect mobility
-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)
how does the “Ionic Strength of Buffers” affect mobility
-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
how does the “Effect of Increased Ionic Strength” affect mobility
-greater the ionic strength the greater the resistance to movement (decreased migration)
-cleaner fractions increased resolution
-increased heat production can denature heat labile proteins
how does the “Electroendosmosis” affect mobility
- overall solvent movement
-movement of buffer ions and buffer solvent relative to fixed support medium
why would you order SPE
-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
What is SPE
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
how to fix and stain after electrophoresis
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
Quantitation in PE
-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
Densitometric scan: Serum Proteins what are the peaks seen as
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
What is albumin
-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
What is alpha 1 globulin
-alpha-1-antitrypsin, alpha-1-acid glycoprotein, and alpha-1-lipoprotein
-increases indicate active tissue damage like inflammation, malignancy and post op conditions
What is alpha 2 globulin
-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
Nephrotic syndrome
decrease in albumin
decrease in gamma globulins
increase in alpha 2 globulins
What is beta globulin
made up of
transferrin
beta lipoprotein
complement C3 (heat labile and decreases if stored at room temp)
-visible changes here are uncommon
what gamma globulins
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
What will you have if you have an increase in a single globulin out of the three
Multiple myeloma
Waldenstrom’s macroglobulinemia
Bench-Jones proteinemia
What will be seen on monoclonal gammopathies
-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
what is seen in cirrhosis of liver
decrease of albumin
increase in gamma
bridging between beta and gamma
what will you see in polyclonal gammopathy
=hypergammaglobulinemia
-increase of all gamma globulins
-decreased albumin
due to chronic infection
liver disease
burn
autoimmune disease
what will you see in liver disease
decreased albumin
increased gamma globulins (polyclonal)
beta gamma bridging
what is hypogammaglobulinemia
decreased or absent gamma globulin due to congitenal disorder (IgA or IgG def) and immunosuppressive therapy
what will you see in viral hepatitis
decrease in albumin
increase in gamma globulins
What will you see in chronic inflammation
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
how can you apply electrophoresis
spinal fluid proteins
-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
What are OLIG banding in spinal fluid proteins
-seen in gamma fraction in pt with MS
-can be seen very early and will be in the bottom of gel toward you
how can you apply electrophoresis
isoenzymes
, 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
how can you apply electrophoresis
isoenzymes CK
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
how can you apply electrophoresis
Lipoproteins
-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
What to watch for in electrophoresis
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
What to watch for in electrophoresis
band irregularities
-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
What to watch for in electrophoresis
buffers
-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
What will hemolysed samples be seen as
-increase in b2 area where the hemoglobin migrates or a band between a2 and b2 from the Hb-HAP complex
What will fibrinogen in samples be seen as
show near application point which is why we use serum samples to avoid this extra band
why will you see a spilt albumin peak or a wide peak
wide - drug that is albumin bound
split - benign condition called bisalbuminemia
What will you have to do before putting the sample in the gel
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
What will you see in Hb electrophoresis
hbA with small HbF and HbA2
the HbA will migrate to beta region
What is HBA1C
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
how will heat affect gets
evaporates solvent from the ends of support medium
-drying will cause “wick up” on both ends of support to replace moisture loss
