Electrophoresis Flashcards
What is electrophoresis?
It is a technique that involves migration of charged solutes in a liquid medium when an electrical field is applied
What type of technique is electrophoresis?
A separation technique
Towards what direction do negatively charged particles move?
Towards the anode (positively charged electrode)
Towards what direction do the positively charged particles move?
Towards the cathode (negatively charged electrode)
Depending on what side chains are present, what type of charge do proteins have?
They are amphoteric (can cat both as cation or anion)
What is electrophoretic mobility?
It is the rate of migration of particles in cm/s per unit of field strength as volts/cm
What is the isoelectric point of a protein?
The pH at which that protein has no net charge
What is the isoelectric point of albumin?
4.9
What range do the isoelectric points of globulins fall?
4.0-7.3
What is the isoelectric point of hemoglobin?
~ 7
What charge does a protein become when buffer pH is above the isoelectric point of that protein?
The protein becomes negative and moves towards the anode
What is the typical pH of the protein electrophoresis buffer?
8.6
How is the carboxyl group of proteins when they’re negatively charged ,owing towards the anode?
The carboxyl group is ionized
What is migration rate of particles?
How quickly particles move toward an electrode.
What factors affect the migration rate of particles?
Net charge of particles (most important factor, bigger the charge, the faster the particle)
Size and shape of molecules (bigger the size, the slower the particle)
Electrical field (more current, faster particle)
Ionic strength of buffer (greater ionic strength, faster particle)
pH of buffer (lower pH of buffer, slower particle)
Viscosity (thicker medium, slower particle)
Temperature (higher temp, faster particle)
What does the net charge of a particle depend on?
On the buffer and the resultant pH set by the buffer
What can too much ionic buffer strength cause?
Increased heat and smearing
Why is care taken with heat sensitive analytes?
High temperature can denature the protein and DNA/RNA
Bands will be smeared or gel will melt
What is the most common type of electrophoresis?
Zone electrophoresis
How does zone electrophoresis work?
ZE is the separation of zones into bands. It separates zones of protein that are distinct from each other.
Name the support media used in zone electrophoresis
Agarose
Polyacrylamide
Cellulose acetate gel
What electrophoresis is mostly seen in the clinical lab and what type is it?
Serum protein electrophoresis
Semi-quantitative
List the electrophoresis instruments
Support medium Buffer Sample Power supply Dye/stain Dryer/scanner
What electrophoresis instrument is crucial for good separation of bands and why?
Buffer. It protects the same place, applies charge to it and carries current
What type of sample is usually used in SPE?
Serum
Whole blood
CSF
Urine
What is done to the finished gel to reveal the bands?
The gel is stained
The intensity of staining is proportional to what?
The amount of protein in the band
Through what scanning is electrophoresis measured?
Densitometry scanning
What is another name for electroendosmosis?
Endosmosis
What is endosmosis?
This is when several gels used in SPE attract positive ions from the buffer and they form a positive ion cloud that moves in opposite direction of the proteins, that is towards the cathode
How does endosmosis affect the move,ent of sample proteins?
The migration of some proteins might be slowed
Some proteins can become immobile
Other proteins are even pushed towards the cathode
Can equally help to achieve better separation of bands
Gamma globulin band will separate more sharply
What is the most negative,y charged protein?
Albumin
What protein migrates closest to the anode?
Albumin
This protein has the highest dye binding capacity and why?
Albumin, because it is the major protein in blood
How rare or common are shifts in albumin?
Increases are rare and seen in dehydration
Decreases are common and seen in alcoholism, liver disease, nephrotic syndrome, burns, myeloma, acute/chronic inflammation, pregnancy
In what condition is albumin levels low?
Pregnancy Liver disease Acute/chronic inflammation Alcoholism Nephrotic syndrome Burns Myeloma
List the a1 globulins
a1 antitrypsin
a1 acid glycoprotein
a1 fetoprotein
HDL
Where is a1 acid gp made and what does it do?
It is made in the liver and transports drugs
What causes emphysema?
A genetic deficiency or mutation of a1 antitrypsin where neutrophils destroy lung tissue via elastase
In what conditions is a1 fetoprotein increased?
Neural tube defects
Liver disease
Germ cell/ liver cancer
In what condition is a1 fetoprotein deceased?
Down syndrome
What enzyme causes neutrophils to destroy lung tissue?
Elastase
List the a2 globulins
Haptoglobin
Ceruloplasmin
a2 macroglobulin
Antithrombin III
What does haptoglobin do?
It binds free Hgb which is toxic to the body
What does ceruloplasmin do?
It binds copper in the body
What is the function of a2 macroglobulin?
Serves as humor defense against pathogenic peptides
What protein keeps blood from clotting abnormally and what class of serum protein does it fall in?
Antithrombin III
It is an a2 protein
List the beta globulins
Transferrin
Free Hgb
b2 microglobulin
LDL, VLDL
What does transferrin do?
It binds free iron in plasma
What does b2 microglobulin do?
It has immune function
How are Ig arranged from the beta region
IgM is closer to beta region
IgA next
IgG next
List the gamma globulins
Ig
CRP
Why do we have to use serum instead if plasma to analyze serum proteins?
Clotting factors in plasma will migrate to the same zone as the proteins thereby blocking accurate reading of the gamma fraction
What is the predominant protein in a2 globulins?
Haptoglobin
What is the predominant protein in beta globulins?
Transferrin
What is gammopathy?
Inflammation due to Ig response in chronic infection, inflammation, disease or autoimmune diseases
Monoclonal gammopathy is seen in what condition
Multiple myeloma
What does chronic liver disease do to albumin levels?
Decreases albumin levels
What dies liver disease do to gamma globulins?
Increases gamma due to chronic or inflammatory state
What condition leads to beta gamma bridging?
Chronic liver disease like cirrhosis
In what gammopathy is TP higher although it is higher than normal in both?
Monoclonal gammopathy
How does nephrotic syndrome affect proteins?
Decreased albumin levels since they’re lost in kidneys resulting in elevated a2 and beta globulins
What is agammaglobulinemia?
This is decreased Ig production due to conditions like HIV/AIDS or drug induction form cancer chemotherapy or anti-rejection transplant drugs
Identify the condition. Normal TP Low albumin Normal a1 Normal a2 beta levels bridging High Ig
Liver disease
Identify the condition Low TP Low albumin Normal a1 High a2 High beta Normal Ig
Nephrotic syndrome
What buffer is used in Hgb electrophoresis?
Wither acid or alkaline pH buffer depending on the type of Hgb isolated
How is capillary electrophoresis different from others?
It uses a very small bore capillary tube with mobile chamber carrier matrix a as the electrophoretic chamber
What type of electrophoresis allows heat to dissipate quickly so can use extraordinarily high voltages of up to 30000 V?
Capillary electrophoresis
