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CC LAB > Proteins_ FRACTIONATION, IDENTIFICATION, AND QUANTITATION OF SPECIFIC PROTEINS > Flashcards

Proteins_ FRACTIONATION, IDENTIFICATION, AND QUANTITATION OF SPECIFIC PROTEINS Flashcards

1
Q

• Globulins are separated from albumin by salting out, using sodium salt to precipitate globulins

• Globulin: precipitate
• Albumin: supernatant fluid (measured)

A

SALT FRACTIONATION

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2
Q

SALT FRACTIONATION

• Globulins are separated from albumin by salting out, using_____ to precipitate_____

• precipitate
• supernatant fluid (measured)

A

sodium salt\ globulins

Globulin
Albumin

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3
Q

DYE-BINDING (FOR ALBUMIN)

4 dyes

A

• Methyl orange
• 2,4’-hydroxyazobenzene-benzoic acid (HABA)
• Bromocresol Green (BCG)
• Bromocresol Purple (BCP)

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4
Q

ALBUMIN

•procedures: most widely used methods for determining
Albumin.

• pH of the solution is adjusted so that albumin is_____ charged.

• Albumin is attracted to and binds to______ dye

• Amount of albumin = absorbance of the______

A

Dye-binding

positively

anionic dye

albumin-dye complex

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5
Q

DYE-BINDING (FOR ALBUMIN)

• Nonspecific for albumin
• Beta-lipoproteins, alpha-1 and alpha-2 globulins also bind to dye

A

Methyl Orange

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6
Q

DYE-BINDING (FOR ALBUMIN)
Methyl Orange
• Nonspecific for_____
• (3) also bind to dye

A

albumin

Beta-lipoproteins, alpha-1 and alpha-2 globulins

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7
Q

DYE-BINDING (FOR ALBUMIN)

• Has low sensitivity but more specific for albumin
• Salicylates, penicillin, conjugated bilirubin, and sulfonamides - interfere with the binding of albumin to HABA

A

2,4’-hydroxyazobenzene-benzoic acid (HABA)

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8
Q

DYE-BINDING (FOR ALBUMIN)
2,4’-hydroxyazobenzene-benzoic acid (HABA)

• Has low_____ but more_____ for albumin
• (4) - interfere with the binding of albumin to HABA

A

sensitivity\ specific

Salicylates, penicillin, conjugated bilirubin, and sulfonamides

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9
Q

DYE-BINDING (FOR ALBUMIN)

• Has been reported to overestimate low albumin values (nephrotic syndrome or end-stage renal disease)

A

Bromocresol Green (BCG)

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10
Q

DYE-BINDING (FOR ALBUMIN)
Bromocresol Green (BCG)

• Hemoglobin binds to BCG (for every 100 mg/dL of Hgb, albumin increased by_____)

• Has been reported to overestimate low albumin values (nephrotic syndrome or end-stage renal disease)

A

0.1 g/dL

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11
Q

DYE-BINDING (FOR ALBUMIN)

• Alternate dye, binding specifically to albumin

• Not subject to interferences, precise, exhibits excellent correlation with immunodiffusion reference methods

A

Bromocresol Purple (BCP)

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12
Q

DYE-BINDING (FOR ALBUMIN)

• In renal insufficiency, this method underestimates serum albumin

• Bilirubin interferes with this binding to Albumin

A

Bromocresol Purple (BCP)

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13
Q

DYE-BINDING (FOR ALBUMIN)
Bromocresol Purple (BCP)

• Alternate dye, binding specifically to____

• Not subject to_____, precise, exhibits excellent correlation with immunodiffusion reference methods

• In renal insufficiency, BCP method underestimates_____

•_____ interferes with BCP binding to Albumin

A

albumin

interferences

serum albumin

Bilirubin

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14
Q

TOTAL GLOBULINS
• Albumin can be calculated by

A

subtraction of the globulin from Total Protein

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15
Q

• Total Globulin level:

direct colorimetric method using_____

A

glyoxylic acid

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16
Q

TOTAL GLOBULINS

•______, in the presence of Cu2+ and in an acid medium (acetic acid and H,SO4), condenses with____ found in globulins to produce_____ color.

A

Glyoxylic acid

tryptophan

purple

17
Q

pI of Albumin

A

Negative (-) so sa Anode siya magdiretso (+)

18
Q

Alpha 1

A

a1-Antitrypsin
a1-Fetoprotein
a1-Acid glycoprotein
a1-Lipoprotein
a1-Antichymotrypsin
Inter-a-trypsin inhibitor
Gc-globulin

19
Q

Alpha 2

A

HAPTOGLOBINS
Ceruloplasmin
a2-Macroglobulin

20
Q

Beta

A

Pre-3 lipoprotein
Transferrin
Hemopexin
B-Lipoprotein
ß2-Microglobulin (B2M)
Complement
Fibrinogen
CRP

21
Q

Gamma

A

IgG
IgA
IgM
IgD
lgE

22
Q

• Separates proteins on the basis of their electric charge densities.

A

ELECTROPHORESIS

23
Q

• Protein will move according to their____ determined by pH of a surrounding buffer.

• Direction of movement depends on___

• Speed of migration can be estimated from the difference between the____ of the protein and the___ of buffer.

A

ELECTROPHORESIS

density

charge

pl\ ph

24
Q

ELECTROPHORESIS

•_____ depends on electric field strength, size, and shape of molecule, temperature and characteristics of buffer

A

Velocity

25
Q

ELECTROPHORESIS

•support media

A

Cellulose acetate or agarose gel

26
Q

PRINCIPLE
Globulins are precipitated in high salt concentrations: albumin in supernatant is quantitated by biuret reaction

COMMENT
Labor intensive

A

Salt precipitation

27
Q

Albumin binds to dye; causes shift in
absorption maximum

Nonspecific for albumin

A

Methyl orange

28
Q

Albumin binds to dye; causes shift in
absorption maximum

Many interference (salicylates, bilirubin)

A

HABA [2,4’-hydroxyazobenzene-
benzoic acid

29
Q

Albumin binds to dye; causes shift in
absorption maximum

Sensitive; overestimates low albumin levels; most commonly used dye

A

BCG (bromocresol areen)

30
Q

Albumin binds to dve: causes shift in
absorption maximum

Specific, sensitive, precise

A

BCP (bromocresol purple)

31
Q

Proteins separated based on electric charge

Accurate; gives overview of relative changes in different protein fractions

A

Electrophoresis

CC LAB (46 decks)

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