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Clinical Chem I > Electrophoresis > Flashcards

Electrophoresis Flashcards

1
Q

What is electrophoresis?

A

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

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2
Q

What type of technique is electrophoresis?

A

A separation technique

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3
Q

Towards what direction do negatively charged particles move?

A

Towards the anode (positively charged electrode)

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4
Q

Towards what direction do the positively charged particles move?

A

Towards the cathode (negatively charged electrode)

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5
Q

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

A

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

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6
Q

What is electrophoretic mobility?

A

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

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7
Q

What is the isoelectric point of a protein?

A

The pH at which that protein has no net charge

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8
Q

What is the isoelectric point of albumin?

A

4.9

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9
Q

What range do the isoelectric points of globulins fall?

A

4.0-7.3

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10
Q

What is the isoelectric point of hemoglobin?

A

~ 7

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11
Q

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

A

The protein becomes negative and moves towards the anode

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12
Q

What is the typical pH of the protein electrophoresis buffer?

A

8.6

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13
Q

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

A

The carboxyl group is ionized

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14
Q

What is migration rate of particles?

A

How quickly particles move toward an electrode.

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15
Q

What factors affect the migration rate of particles?

A

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)

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16
Q

What does the net charge of a particle depend on?

A

On the buffer and the resultant pH set by the buffer

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17
Q

What can too much ionic buffer strength cause?

A

Increased heat and smearing

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18
Q

Why is care taken with heat sensitive analytes?

A

High temperature can denature the protein and DNA/RNA

Bands will be smeared or gel will melt

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19
Q

What is the most common type of electrophoresis?

A

Zone electrophoresis

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20
Q

How does zone electrophoresis work?

A

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

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21
Q

Name the support media used in zone electrophoresis

A

Agarose
Polyacrylamide
Cellulose acetate gel

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22
Q

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

A

Serum protein electrophoresis

Semi-quantitative

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23
Q

List the electrophoresis instruments

A 
Support medium 
Buffer
Sample
Power supply
Dye/stain
Dryer/scanner
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24
Q

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

A

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

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25
Q

What type of sample is usually used in SPE?

A

Serum
Whole blood
CSF
Urine

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26
Q

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

A

The gel is stained

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27
Q

The intensity of staining is proportional to what?

A

The amount of protein in the band

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28
Q

Through what scanning is electrophoresis measured?

A

Densitometry scanning

29
Q

What is another name for electroendosmosis?

A

Endosmosis

30
Q

What is endosmosis?

A

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

31
Q

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

A

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

32
Q

What is the most negative,y charged protein?

A

Albumin

33
Q

What protein migrates closest to the anode?

A

Albumin

34
Q

This protein has the highest dye binding capacity and why?

A

Albumin, because it is the major protein in blood

35
Q

How rare or common are shifts in albumin?

A

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

36
Q

In what condition is albumin levels low?

A 
Pregnancy 
Liver disease
Acute/chronic inflammation 
Alcoholism
Nephrotic syndrome 
Burns
Myeloma
37
Q

List the a1 globulins

A

a1 antitrypsin
a1 acid glycoprotein
a1 fetoprotein
HDL

38
Q

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

A

It is made in the liver and transports drugs

39
Q

What causes emphysema?

A

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

40
Q

In what conditions is a1 fetoprotein increased?

A

Neural tube defects
Liver disease
Germ cell/ liver cancer

41
Q

In what condition is a1 fetoprotein deceased?

A

Down syndrome

42
Q

What enzyme causes neutrophils to destroy lung tissue?

A

Elastase

43
Q

List the a2 globulins

A

Haptoglobin
Ceruloplasmin
a2 macroglobulin
Antithrombin III

44
Q

What does haptoglobin do?

A

It binds free Hgb which is toxic to the body

45
Q

What does ceruloplasmin do?

A

It binds copper in the body

46
Q

What is the function of a2 macroglobulin?

A

Serves as humor defense against pathogenic peptides

47
Q

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

A

Antithrombin III

It is an a2 protein

48
Q

List the beta globulins

A

Transferrin
Free Hgb
b2 microglobulin
LDL, VLDL

49
Q

What does transferrin do?

A

It binds free iron in plasma

50
Q

What does b2 microglobulin do?

A

It has immune function

51
Q

How are Ig arranged from the beta region

A

IgM is closer to beta region
IgA next
IgG next

52
Q

List the gamma globulins

A

Ig

CRP

53
Q

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

A

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

54
Q

What is the predominant protein in a2 globulins?

A

Haptoglobin

55
Q

What is the predominant protein in beta globulins?

A

Transferrin

56
Q

What is gammopathy?

A

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

57
Q

Monoclonal gammopathy is seen in what condition

A

Multiple myeloma

58
Q

What does chronic liver disease do to albumin levels?

A

Decreases albumin levels

59
Q

What dies liver disease do to gamma globulins?

A

Increases gamma due to chronic or inflammatory state

60
Q

What condition leads to beta gamma bridging?

A

Chronic liver disease like cirrhosis

61
Q

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

A

Monoclonal gammopathy

62
Q

How does nephrotic syndrome affect proteins?

A

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

63
Q

What is agammaglobulinemia?

A

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

64
Q 
Identify the condition. 
Normal TP
Low albumin 
Normal a1
Normal a2
beta levels bridging
High Ig
A

Liver disease

65
Q 
Identify the condition 
Low TP
Low albumin
Normal a1
High a2
High beta
Normal Ig
A

Nephrotic syndrome

66
Q

What buffer is used in Hgb electrophoresis?

A

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

67
Q

How is capillary electrophoresis different from others?

A

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

68
Q

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

A

Capillary electrophoresis

Clinical Chem I (17 decks)

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