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Electrophoresis Flashcards

1
Q

Electrophoresis definition

A

defined as the movement or
migration of charged particles in an electric
field

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2
Q

Electrophoresis is used to

A

Routinely used to separate serum proteins,
hemoglobin, enzymes, lipoproteins,
immunoglobulins, urine proteins, CSF
proteins, and other body fluid proteins

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3
Q

Electrophoresis is responsible in advances in what

A

Most of the remarkable advances in molecular
biology over the past few decades would have
been impossible without electrophoretic methods

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4
Q

Types of electrophoresis

A

Free electrophoresis and Zone electrophoresis

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5
Q

Free electrophoresis types

A

-Micro
Electrophoresis
-Moving
Boundary

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6
Q

Zone electrophoresis types

A

-Paper
Electrophoresis
Cellulose
-Acetate
Electrophoresis
-Gel
Electrophoresis

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7
Q

Iontophoresis is

A

he migration of small ions

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8
Q

Zone electrophoresis is

A

migration of charged
macromolecules in a porous support medium.

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9
Q

Electrophoresis system components

A

§ Charged particles
§ Support medium
§ Buffer
§ Electrical Power Source
§ Detecting system

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10
Q

Proteins are what and contain what

A

Proteins are amphoteric, containing both acidic and basic groups

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11
Q

Proteins charge is positive at

A

Low pH

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12
Q

Proteins zero (isoelectric)

A

at a particular higher pH

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13
Q

Proteins negative at

A

Alkaline pH

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14
Q

Electrophoretic mobility is proportional to

The effective mobility of a protein is

A

§ Electrophoretic mobility is directly proportional to the size of the
charge of the particle
§ The effective mobility of a protein is a function of pH

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15
Q

Support media types

A

§ AGAROSE GEL—most often used in clinical settings
§ POLYACRYLAMIDE GEL
§ STARCH GEL
§ CELLULOSE ACETATE
§ PAPER

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16
Q

Buffer functions

A

§ The buffer serves several functions:
§ Carries the applied current
§ Establishes and maintains a constant pH
§ Determines the electrical charge on the solute

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17
Q

Buffers

Protein separator buffers

A

§ Barbital buffers
§ Tris-boric acid-EDTA

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18
Q

Nucleic acid separation buffers

A

§ Buffers containing EDTA and Tris-acetate, Tris-borate, or Tris-
phosphate

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19
Q

Electrophoresis is conducted for a

A

§ Electrophoresis is conducted for a determined length of time
under conditions of either constant voltage or constant current.

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20
Q

Separated fractions are

A

§ Separated fractions are stained for visualization and densitometry

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21
Q

Numerous stains have been used and depend on

A

§ Numerous stains have been used, dependent upon the particular
analyte

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22
Q

Protein stains

A

§ Proteins:
§ Ponseau S Silver stain
§ Bromcresol blue Stains All
§ Light green SF Amido Black 10B
§ Coumassie blue R250 Colloidal Gold

23
Q

Lipoprotein stain

A

§ Sudan black B
§ Oil Red O
§ Coumassie brilliant blue R25

24
Q

Glycoprotein stains

A

§ PAS (Periodic-acid-Schiff)
§ Stains-All

25
Nucleic acid stains
§ Stains All § Silver stains § Ethidium bromide § TOTO-1
26
Enzyme stains
§ NADH (fluorescence) § Nitroblue tetrazolium chloride
27
Esterase stains
§ Beta-naphthyl esters and tetrazotized o-dianisidine
28
§ Cholinesterases & Phosphatase stains
§ 1-naphthyl phosphate and fast blue B
29
Factors affecting rate of migration
§ Net charge of the molecule § Size and shape of the molecule § Strength of the electric field § Temperature § Ionic strength of buffer § Wick flow § Electroendosmosis/ (Endosmosis)
30
Reference range of total protein
6.0-8.0g/dL
31
Reference range of Albumin
3.5-5.0g/dL
32
Reference range of a1-globulins
0.1-0.4g/dL
33
Reference range of a2-globulins
.4-1.3g/dL
34
B-globulin reference range
.6-1.3g/dL
35
Reference range of y-globulin
.6-1.5g/dL
36
The acute inflammatory response is
* Immediate response occurs with stress or inflammation caused by infection, injury or surgical trauma
37
Acute inflammatory response effects what values
* normal or ↓ albumin * ↑ α1 and α2 globulins
38
Acute Inflammatory response picture
39
Chronic inflammatory response
40
Liver damage
* Cirrhosis can be caused by chronic alcohol abuse or viral hepatitis
41
Liver damage affects what values
* ↓ albumin * ↓ α1, α2 and β globulins * ↑ Ig A in γ-fraction
42
Liver damage picture
43
Nephrotic syndrome loss of what retention of what
* the kidney damage illustrates the long term loss of lower molecular weight proteins (↓ albumin and IgG – they are filtered in kidney) * retention of higher molecular weight proteins (↑↑ α2-macroglobulin and ↑β- globulin
44
Nephrotic syndrome picture
45
Monoclonal Gammopathy causes Production of blank is associated with Blank can be found in a different what and positions
§ Monoclonal gammapathy is caused by monoclonal proliferation of β-lymphocytal clones. These “altered” β-cells produce an abnormal immunoglobulin paraprotein. § Production of paraprotein is associated with benign monoclonal gammopathy (leucemia) and multiple myeloma. § Paraproteins can be found in a different position: between α-2 and γ-fraction.
46
Monoclonal Gammopathy pictures
47
Urine proteins normal physiology They appear in blank because
* Proteins found in the urine are from the blood; however, urinary proteins can originate from the kidney and urinary tract and from extraneous sources such as the vagina or prostate. * They appear in the urine because they have passed through the renal glomerulus and have not been reabsorbed by the renal tubule
48
CSF proteins Changes are usually do to what What should accompany it mechanism
About 95% of the proteins found in CSF result from active transport of plasma proteins at the blood-CSF barrier. § Changes are usually due to lesions, trauma, vascular damage or infection. § CSF protein electrophoresis should also accompany serum electrophoresis
49
Other Applications of electrophoresis used in special chem
§ Isoenzyme Electrophoresis § CK, LD, ALP & others § Lipid Electrophoresis § Hemoglobin Electrophoresis § Glycoprotein Electrophoresis § Nucleic Acid Electrophoresis
50
Hemoglobin and Heme derivatives Migration is from In the Blank, Hb is In blank, Hb is
§ Migration is from cathode to anode. § In the Cellulose Acetate (pH 8.4), Hb is negatively charged. § In Citrate Agar (pH 6.0), Hb is positively charged.
51
Cellulose acetate and Citrate agar picture
52
Immunoelectrophoresis
§ Antigen is added to the well and electrophoresis is carried out. Under the electric field, the charged antigens get separated based on size and charge. § Following the separation of the antigens, the antisera is added to the trough and incubated overnight. During that time the antigen-antibody reaction causes a precipitation where the equivalence point is met
53
Rocket electrophoresis
§ Rocket Electrophoresis § Electroimmunodiffusion § Type of immunoassay wherein a single concentration gradient is established for the antigen, and a voltage is applied, which drives the antigen from the application well into a homogeneous suspension of antibody in the gel. § The height of the rocket shaped precipitin line is proportional to the antigen concentration.
54
Electrophoresis techniques The tests specimen may be The primary use of IFE is
§ The tests specimen may be serum, urine, CSF, or other body fluids § The primary use of IFE in clinical laboratories is for the characterization of monoclonal Igs

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