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CC1 LECTURE DEAN > ELECTROPHORESIS > Flashcards

ELECTROPHORESIS Flashcards

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1
Q

Seen in diagnostic laboratories

A

ELECTROPHORESIS

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2
Q

migration of charged solutes or particles in a solid medium under the influence of an electrical field/current.

A

ELECTROPHORESIS

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3
Q

Two types of electrophoresis:

A

Zone electrophoresis
Iontophoresis

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4
Q

– separation of macromolecules on an electromagnetic field or support medium under the influence of electric current

A

Zone electrophoresis

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5
Q

– separation of small particles such as ions or electrolytes (Ex. Separation of Calcium ions in a substance)

A

Iontophoresis

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6
Q

– most important macromolecule

A

Protein

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7
Q

Zwitterion

A

AMPHOLYTE

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8
Q

amphoteric pH (can act as either acid+cathode or base-anode) depending upon the support medium where they are found

A

AMPHOLYTE (Zwitterion)

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9
Q

takes a positive charge (binds protons) at an acidic pH and migrates toward the cathode

A

Cathode

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10
Q

negative electrode

A

Cathode

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11
Q

it tends to bind with proton = receives a proton = carries a positive charge

A

Cathode

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12
Q

takes a negative charge (loses protons) at an alkaline pH and migrates toward the anode - more common

A

Anode

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13
Q

positive electrode

A

Anode

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14
Q

it tends to donate and give up proton = loses a proton = carries a negative charge

A

Anode

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15
Q

generated from electrophoresis

A

ELECTROPHORETOGRAM

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16
Q

display of protein zones, each one (fraction) sharply separated from neighboring zones on the support medium/material

A

ELECTROPHORETOGRAM

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17
Q

Per column represents one [?] (serum or any biological sample).

A

sample

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18
Q

Different fractions per column represents one [?].

A

protein fraction

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19
Q

The thicker the protein zone, the higher the [?].

A

protien concentration

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20
Q

FACTORS AFFECTING MIGRATION:

A

Net electrical charge of molecule (pH of buffer)
MW of the particle
Strength of the electrical field
Type of support medium

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21
Q

The heavier the protein, the slower it migrates

A

MW of the particle

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22
Q

Ex. Serum protein electrophoresis – smallest protein founf in normal serum: [?], which migrates the fastest

A

Albumin

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23
Q

Constantly maintained

A

Strength of the electrical field

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24
Q

A strip of paper can be moistened using a specific buffer to be used for electrophoresis.
The specific sample subjected to electrophoresis will be applied.
The filter paper can be placed between two glass slides.
Both ends of the filter paper (electrodes) must be in contact with the electrolyte solution found in the two chambers (containers)
Electric current will be applied to facilitate the separation of proteins placed on the paper.

A

PAPER ELECTROPHORESIS

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PAPER ELECTROPHORESIS DRAWBACKS
excessive background failing electrophoresis time may be longer when paper is utilized
26
too many shadows found per fraction instead of very distinct, clearly demarcated zones
excessive background failing
27
Type of support medium 
1. Paper  2. Starch (starch gel)  3. Cellulose acetate 4. Agarose gel  5. Polyacrylamide Gel
28
medium is prepared before electrophoresis
Starch (starch gel) 
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greater resolution
Cellulose acetate
30
faster & permanent 
Cellulose acetate
31
distinct zones are separated
Cellulose acetate
32
most commonly used in electrophoresis
Cellulose acetate
33
most commonly used in electrophoresis
Agarose gel 
34
High Resolution Electrophoresis
Polyacrylamide Gel
35
most commonly used medium in isoelectric focusing
Polyacrylamide Gel
36
- 45 minutes to 1 hour
AGAROSE GEL ELECTROPHORESIS
37
1. Mix the agarose with the buffer of choice 2. Heated in a microwave until such time that the buffer agarose is completely dissolved 3. The agarose mixture will be poured into the mould 4 . At one end of the mould, a comb is inserted
AGAROSE GEL ELECTROPHORESIS
38
TYPES OF BUFFER
1. Barbital (pH 8.6)  2. Citric Acid – PO4 (pH 6.0-6.2)  3. Tris-Boric EDTA (pH 8.4-8.6) 
39
Buffer: Citric Acid Solid support: Agarose Gel
Citrate-agar electrophoresis Citrate-phosphate-agar electrophoresis
40
Buffer: Alkaline solution Solid support: Cellulose acetate
Tris-Boric EDTA (pH 8.4-8.6)  Tris-EDTA-Boric
41
- for preservation
FIXATIVE
42
TYPES OF FIXATIVE
1. Methanol  2. Cyclohexanol  3. Acetic acid  4. Dioxane 
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- to allow visualization of the protein zones that have been separated
STAINS
44
Protein stains:
- Amido black or Naphthol blue black - Bromphenol blue - Coomasie Brilliant blue - Nigrosin  - Ponceau S dye 
45
Isoenzymes: 
- Nitrotetrazoleum Blue 
46
Lipoproteins (HDL, LDL, VLDL): 
- Fat Red 7B (Sudan Red)  - Oil Red O  - Sudan Black B 
47
are separated based on their isoelectric pH
Proteins
48
are made up of amino acids
Proteins
49
is determined by the type of amino acid that predominates in their structure on the basis of acidity (neutral, acidic, basic)
isoelectric pH
50
An electrophoretic method in w/c proteins are separated on the basis of their
pI (isoelectric pH) 
51
Makes use of the property of proteins that their net charges are determined by the [?] of their local environment 
pH
52
net electrical charges 
(+), (-) or (0)
53
If the # of acidic groups (in protein) exceeds # of basic groups, the pI of the protein will be at a [?] pH (ACIDIC PROTEIN) 
low
54
If the basic group > acidic groups, pI will be [?] (BASIC PROTEIN)
high
55
Proteins show considerable variation in pI, but pI values fall in the range pH [?] (many having pIs between pH 4-7) 
3-12
56
REQUIREMENTS FOR ISOELECTRIC FOCUSING
1. Establishing pH gradient 2. Gel for Isoelectric Focusing
57
1. Establishing pH gradient
a. Carrier Ampholytes (Amphoteric electrolytes)  b. Acrylamide buffers 
58
2. Gel for Isoelectric Focusing
a. Polyacrylamide Gel
59
most commonly used in isoelectric focusing to maintain a pH gradient
Acrylamide buffers
60
- Mixtures of molecules containing multiple aliphatic amino & carboxylate groups (buffer molecules) 
a. Carrier Ampholytes (Amphoteric electrolytes) 
61
- Included directly in IEF gels 
a. Carrier Ampholytes (Amphoteric electrolytes) 
62
- Derivatives of Acrylamide containing both reactive double bonds & buffering groups 
b. Acrylamide buffers
63
- Covalently incorporated in PAG at the time of casting 
b. Acrylamide buffers
64
- polymerized with an initiator system including Riboflavin (initiator) for photo-polymerization
a. Polyacrylamide Gel
65
- large-pore convective matrices 
a. Polyacrylamide Gel
66
1. A stable pH gradient is established in the gel after application of an electric field. 2. A protein solution is added and an electrical field is reapplied. 3. After staining, proteins are to be distributed along a pH gradient, according to pH values
67
Addition of carrier ampholyte or acrylamide buffers into the support medium
1. A stable pH gradient is established in the gel after application of an electric field.
68
Allow the support medium to solidify = POLYACRYLAMIDE GEL
2. A protein solution is added and an electrical field is reapplied.
69
Available locations for electric current to flow through the medium.
2. A protein solution is added and an electrical field is reapplied.
70
On the surface of the solidified polyacrylamide gel, place the protein solution.
2. A protein solution is added and an electrical field is reapplied.
71
Followed by the application of electric current.
2. A protein solution is added and an electrical field is reapplied.
72
After appropriate separation time;
2. A protein solution is added and an electrical field is reapplied.
73
Stains are used to visualize protein zones that have been separated
3. After staning, proteins are to be distributed along a pH gradient, according to pH values
74
AUTOMATION ADVANTAGES:  1. [?] results  2. Increase in the [?] performed  3. Saves [?]  4. Eliminates the need for [?] 5. [?]  6 . Errors in [?] are reduced  7. Better [?]
Rapid number of tests time and effort personnel increase  Economical calculations & transcriptions precision & accuracy 
75
- introductory of the first automated analyzer by Technicon
1957
76
: sequential batch analyzer capable of providing single test result on approximately 40 samples per hour
Continuous flow
77
- Technicon instruments w/c were next developed 
Simultaneous Multiple Analyzer (SMA)
78
With multiple channels (for diff. tests) 
Simultaneous Multiple Analyzer (SMA)
79
6-12 test results simultaneously at the rate of 360 - 720 tests per hour 
Simultaneous Multiple Analyzer (SMA)
80
- specialty area w/ rapidly developing arsenal of analyzers 
IMMUNOCHEMISTRY
81
Immunological techniques (application of antigen antibody reaction) for assaying drugs, specific proteins, tumor markers & hormones 
Fluorescence Polarization Immunoassay  Nephelometry Chemiluminescent Detection 
82
BASIC APPROACHES OF AUTOMATED ANALYZERS
I. CONTINUOUS - FLOW II. CENTRIFUGAL ANALYSIS III. DISCRETE ANALYSIS
83
- Liquids (reagents, diluents & samples) are pumped through a system of continuous tubing 
I. CONTINUOUS - FLOW
84
- Samples are introduced in a sequential manner, following each other through the same network 
I. CONTINUOUS - FLOW
85
- Batch analysis can be used (e.g. large # of specimen in one run) 
I. CONTINUOUS - FLOW
86
- More sophisticated continuous flow anayzers  Use parallel single channels to run multiple tests on each sample (e.g. SMA & SMAC) 
I. CONTINUOUS - FLOW
87
- Major drawbacks: significant carry-over problems & wasteful use of continuously flowing reagents 
I. CONTINUOUS - FLOW
88
- More sophisticated continuous flow anayzers 
I. CONTINUOUS - FLOW
89
- Use parallel single channels to run multiple tests on each sample (e.g. SMA & SMAC) 
I. CONTINUOUS - FLOW
90
- Uses the force generated by centrifugation to transfer & then contain liquids in separate cuvets for measurement 
II. CENTRIFUGAL ANALYSIS
91
- Capable of running multiple samples, one test ata time, ina batch 
II. CENTRIFUGAL ANALYSIS
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- MAJOR ADV.: batch analysis (e.g. COBAS - Bio by Roche Diagnostics) 
II. CENTRIFUGAL ANALYSIS
93
- Most popular & versatile 
III. DISCRETE ANALYSIS
94
- Separation of each sample & accompanying reagents in a separate container 
III. DISCRETE ANALYSIS
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- Capability of running multiple tests one sample at a time OR multiple samples one test ata time  - Random access, stat capabilities 
III. DISCRETE ANALYSIS

CC1 LECTURE DEAN (14 decks)

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