Proteins_ FRACTIONATION, IDENTIFICATION, AND QUANTITATION OF SPECIFIC PROTEINS

Question 1

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

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

Answer 1

SALT FRACTIONATION

Question 2

SALT FRACTIONATION

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

• precipitate
• supernatant fluid (measured)

Answer 2

sodium salt\ globulins

Globulin
Albumin

Question 3

DYE-BINDING (FOR ALBUMIN)

4 dyes

Answer 3

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

Question 4

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______

Answer 4

Dye-binding

positively

anionic dye

albumin-dye complex

Question 5

DYE-BINDING (FOR ALBUMIN)

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

Answer 5

Methyl Orange

Question 6

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

Answer 6

albumin

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

Question 7

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

Answer 7

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

Question 8

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

Answer 8

sensitivity\ specific

Salicylates, penicillin, conjugated bilirubin, and sulfonamides

Question 9

DYE-BINDING (FOR ALBUMIN)

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

Answer 9

Bromocresol Green (BCG)

Question 10

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)

Answer 10

0.1 g/dL

Question 11

DYE-BINDING (FOR ALBUMIN)

• Alternate dye, binding specifically to albumin

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

Answer 11

Bromocresol Purple (BCP)

Question 12

DYE-BINDING (FOR ALBUMIN)

• In renal insufficiency, this method underestimates serum albumin

• Bilirubin interferes with this binding to Albumin

Answer 12

Bromocresol Purple (BCP)

Question 13

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

Answer 13

albumin

interferences

serum albumin

Bilirubin

Question 14

TOTAL GLOBULINS
• Albumin can be calculated by

Answer 14

subtraction of the globulin from Total Protein

Question 15

• Total Globulin level:

direct colorimetric method using_____

Answer 15

glyoxylic acid

Question 16

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.

Answer 16

Glyoxylic acid

tryptophan

purple

Question 17

pI of Albumin

Answer 17

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

Question 18

Alpha 1

Answer 18

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

Question 19

Alpha 2

Answer 19

HAPTOGLOBINS
Ceruloplasmin
a2-Macroglobulin

Question 20

Beta

Answer 20

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

Question 21

Gamma

Answer 21

IgG
IgA
IgM
IgD
lgE

Question 22

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

Answer 22

ELECTROPHORESIS

Question 23

• 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.

Answer 23

ELECTROPHORESIS

density

charge

pl\ ph

Question 24

ELECTROPHORESIS

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

Answer 24

Velocity

Question 25

ELECTROPHORESIS

•support media

Answer 25

Cellulose acetate or agarose gel

Question 26

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

COMMENT
Labor intensive

Answer 26

Salt precipitation

Question 27

Albumin binds to dye; causes shift in
absorption maximum

Nonspecific for albumin

Answer 27

Methyl orange

Question 28

Albumin binds to dye; causes shift in
absorption maximum

Many interference (salicylates, bilirubin)

Answer 28

HABA [2,4’-hydroxyazobenzene-
benzoic acid

Question 29

Albumin binds to dye; causes shift in
absorption maximum

Sensitive; overestimates low albumin levels; most commonly used dye

Answer 29

BCG (bromocresol areen)

Question 30

Albumin binds to dve: causes shift in
absorption maximum

Specific, sensitive, precise

Answer 30

BCP (bromocresol purple)

Question 31

Proteins separated based on electric charge

Accurate; gives overview of relative changes in different protein fractions

Answer 31

Electrophoresis