Electrophoresis

Question 1

What is electrophoresis?

Answer 1

It is a technique that involves migration of charged solutes in a liquid medium when an electrical field is applied

Question 2

What type of technique is electrophoresis?

Answer 2

A separation technique

Question 3

Towards what direction do negatively charged particles move?

Answer 3

Towards the anode (positively charged electrode)

Question 4

Towards what direction do the positively charged particles move?

Answer 4

Towards the cathode (negatively charged electrode)

Question 5

Depending on what side chains are present, what type of charge do proteins have?

Answer 5

They are amphoteric (can cat both as cation or anion)

Question 6

What is electrophoretic mobility?

Answer 6

It is the rate of migration of particles in cm/s per unit of field strength as volts/cm

Question 7

What is the isoelectric point of a protein?

Answer 7

The pH at which that protein has no net charge

Question 8

What is the isoelectric point of albumin?

Answer 8

4.9

Question 9

What range do the isoelectric points of globulins fall?

Answer 9

4.0-7.3

Question 10

What is the isoelectric point of hemoglobin?

Answer 10

~ 7

Question 11

What charge does a protein become when buffer pH is above the isoelectric point of that protein?

Answer 11

The protein becomes negative and moves towards the anode

Question 12

What is the typical pH of the protein electrophoresis buffer?

Answer 12

8.6

Question 13

How is the carboxyl group of proteins when they’re negatively charged ,owing towards the anode?

Answer 13

The carboxyl group is ionized

Question 14

What is migration rate of particles?

Answer 14

How quickly particles move toward an electrode.

Question 15

What factors affect the migration rate of particles?

Answer 15

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)

Question 16

What does the net charge of a particle depend on?

Answer 16

On the buffer and the resultant pH set by the buffer

Question 17

What can too much ionic buffer strength cause?

Answer 17

Increased heat and smearing

Question 18

Why is care taken with heat sensitive analytes?

Answer 18

High temperature can denature the protein and DNA/RNA

Bands will be smeared or gel will melt

Question 19

What is the most common type of electrophoresis?

Answer 19

Zone electrophoresis

Question 20

How does zone electrophoresis work?

Answer 20

ZE is the separation of zones into bands. It separates zones of protein that are distinct from each other.

Question 21

Name the support media used in zone electrophoresis

Answer 21

Agarose
Polyacrylamide
Cellulose acetate gel

Question 22

What electrophoresis is mostly seen in the clinical lab and what type is it?

Answer 22

Serum protein electrophoresis

Semi-quantitative

Question 23

List the electrophoresis instruments

Answer 23

Support medium 
Buffer
Sample
Power supply
Dye/stain
Dryer/scanner

Question 24

What electrophoresis instrument is crucial for good separation of bands and why?

Answer 24

Buffer. It protects the same place, applies charge to it and carries current

Question 25

What type of sample is usually used in SPE?

Answer 25

Serum Whole blood CSF Urine

Question 26

What is done to the finished gel to reveal the bands?

Answer 26

The gel is stained

Question 27

The intensity of staining is proportional to what?

Answer 27

The amount of protein in the band

Question 28

Through what scanning is electrophoresis measured?

Answer 28

Densitometry scanning

Question 29

What is another name for electroendosmosis?

Answer 29

Endosmosis

Question 30

What is endosmosis?

Answer 30

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

Question 31

How does endosmosis affect the move,ent of sample proteins?

Answer 31

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

Question 32

What is the most negative,y charged protein?

Answer 32

Albumin

Question 33

What protein migrates closest to the anode?

Answer 33

Albumin

Question 34

This protein has the highest dye binding capacity and why?

Answer 34

Albumin, because it is the major protein in blood

Question 35

How rare or common are shifts in albumin?

Answer 35

Increases are rare and seen in dehydration Decreases are common and seen in alcoholism, liver disease, nephrotic syndrome, burns, myeloma, acute/chronic inflammation, pregnancy

Question 36

In what condition is albumin levels low?

Answer 36

``` Pregnancy Liver disease Acute/chronic inflammation Alcoholism Nephrotic syndrome Burns Myeloma ```

Question 37

List the a1 globulins

Answer 37

a1 antitrypsin a1 acid glycoprotein a1 fetoprotein HDL

Question 38

Where is a1 acid gp made and what does it do?

Answer 38

It is made in the liver and transports drugs

Question 39

What causes emphysema?

Answer 39

A genetic deficiency or mutation of a1 antitrypsin where neutrophils destroy lung tissue via elastase

Question 40

In what conditions is a1 fetoprotein increased?

Answer 40

Neural tube defects Liver disease Germ cell/ liver cancer

Question 41

In what condition is a1 fetoprotein deceased?

Answer 41

Down syndrome

Question 42

What enzyme causes neutrophils to destroy lung tissue?

Answer 42

Elastase

Question 43

List the a2 globulins

Answer 43

Haptoglobin Ceruloplasmin a2 macroglobulin Antithrombin III

Question 44

What does haptoglobin do?

Answer 44

It binds free Hgb which is toxic to the body

Question 45

What does ceruloplasmin do?

Answer 45

It binds copper in the body

Question 46

What is the function of a2 macroglobulin?

Answer 46

Serves as humor defense against pathogenic peptides

Question 47

What protein keeps blood from clotting abnormally and what class of serum protein does it fall in?

Answer 47

Antithrombin III | It is an a2 protein

Question 48

List the beta globulins

Answer 48

Transferrin Free Hgb b2 microglobulin LDL, VLDL

Question 49

What does transferrin do?

Answer 49

It binds free iron in plasma

Question 50

What does b2 microglobulin do?

Answer 50

It has immune function

Question 51

How are Ig arranged from the beta region

Answer 51

IgM is closer to beta region IgA next IgG next

Question 52

List the gamma globulins

Answer 52

Ig | CRP

Question 53

Why do we have to use serum instead if plasma to analyze serum proteins?

Answer 53

Clotting factors in plasma will migrate to the same zone as the proteins thereby blocking accurate reading of the gamma fraction

Question 54

What is the predominant protein in a2 globulins?

Answer 54

Haptoglobin

Question 55

What is the predominant protein in beta globulins?

Answer 55

Transferrin

Question 56

What is gammopathy?

Answer 56

Inflammation due to Ig response in chronic infection, inflammation, disease or autoimmune diseases

Question 57

Monoclonal gammopathy is seen in what condition

Answer 57

Multiple myeloma

Question 58

What does chronic liver disease do to albumin levels?

Answer 58

Decreases albumin levels

Question 59

What dies liver disease do to gamma globulins?

Answer 59

Increases gamma due to chronic or inflammatory state

Question 60

What condition leads to beta gamma bridging?

Answer 60

Chronic liver disease like cirrhosis

Question 61

In what gammopathy is TP higher although it is higher than normal in both?

Answer 61

Monoclonal gammopathy

Question 62

How does nephrotic syndrome affect proteins?

Answer 62

Decreased albumin levels since they’re lost in kidneys resulting in elevated a2 and beta globulins

Question 63

What is agammaglobulinemia?

Answer 63

This is decreased Ig production due to conditions like HIV/AIDS or drug induction form cancer chemotherapy or anti-rejection transplant drugs

Question 64

``` Identify the condition. Normal TP Low albumin Normal a1 Normal a2 beta levels bridging High Ig ```

Answer 64

Liver disease

Question 65

``` Identify the condition Low TP Low albumin Normal a1 High a2 High beta Normal Ig ```

Answer 65

Nephrotic syndrome

Question 66

What buffer is used in Hgb electrophoresis?

Answer 66

Wither acid or alkaline pH buffer depending on the type of Hgb isolated

Question 67

How is capillary electrophoresis different from others?

Answer 67

It uses a very small bore capillary tube with mobile chamber carrier matrix a as the electrophoretic chamber

Question 68

What type of electrophoresis allows heat to dissipate quickly so can use extraordinarily high voltages of up to 30000 V?

Answer 68

Capillary electrophoresis