Proteins_ SERUM PROTEIN ELECTROPHORESIS Flashcards by Kyra Catedral

SERUM PROTEIN ELECTROPHORESIS
• Serum are applied close to the_____ end of a support medium (ph_____)

cathode (pH 8.6)

How well did you know this?

Not at all

Perfectly

• All major serum proteins carry a net____ charge at ph 8.6 and migrate toward the____.

negative

anode

How well did you know this?

Not at all

Perfectly

SERUM PROTEIN ELECTROPHORESIS

5 bands:

Albumin
Alpha-1 globulins
Alpha-2 globulins
Beta-globulins
Gamma-globulins

How well did you know this?

Not at all

Perfectly

: travels farthest to the anode

Albumin

How well did you know this?

Not at all

Perfectly

= number of proteins present in that fraction.

Width of the band

How well did you know this?

Not at all

Perfectly

SERUM PROTEIN ELECTROPHORESIS
• After separation, protein fractions are_____ by immersing the support medium in an______

• Next step, proteins are_____.

• Cleared transparent medium is placed in a______ for reading.

fixed - acid solution (acetic acid)

stained

scanning densitometer

How well did you know this?

Not at all

Perfectly

Dyes used in serum protein electrophoresis

Ponceau S
Amido black
Coomassie blue

How well did you know this?

Not at all

Perfectly

• The pattern on the membrane moves past a slit through which light is transmitted to a phototube to record the absorbance of the dye that is bound to each protein fraction.

scanning densitometer

How well did you know this?

Not at all

Perfectly

• Absorbance is recorded on a strip-chart recorder to obtain a pattern of the fraction.

• Compute the area under the absorbance curve for each band and the percentage of total dye that appears in each fraction.

• Concentration is calculated as a percentage of the total protein

• Computation also be made by cutting out the small bands from the membrane and eluting the dye in_____

• Absorbances are added to obtain total absorbance, and the percentage of the total absorbance is calculated

0.1 mol/L NaOH

How well did you know this?

Not at all

Perfectly

Albumin

Percentage and Concentration

53 - 65%

3.5 - 5.0 g/dL

How well did you know this?

Not at all

Perfectly

a-1 globulin

Percentage and Concentration

2.5 - 5%

0.1 - 0.3 g/dL

How well did you know this?

Not at all

Perfectly

a-2 globulins

Percentage and Concentration

7 - 13%

0.6 - 1.0 g/dL

How well did you know this?

Not at all

Perfectly

Beta-globulin

Percentage and Concentration

8 - 14%

0.7 - 1.1 g/dL

How well did you know this?

Not at all

Perfectly

Beta-globulin

Percentage and Concentration

8 - 14%

0.7 - 1.1 g/dL

How well did you know this?

Not at all

Perfectly

Gamma globulin

Percentage and Concentration

12 - 22%

0.8 - 1.6 g/dL

How well did you know this?

Not at all

Perfectly

” gamma spike”

MONOCLONAL IMMUNOGLOBULIN DISEASE

How well did you know this?

Not at all

Perfectly

MONOCLONAL IMMUNOGLOBULIN DISEASE

Gamma spike

How well did you know this?

Not at all

Perfectly

_________ increase in the gamma area, that means there is a_____ so a spike in the Gamma or beta or sometimes in the Alpha 2 region signals the need for examination of immunoglobulins and observed for clinical signs of myelomatosis.

monoclonal increase

monoclonal immunoglobulin disease

How well did you know this?

Not at all

Perfectly

• Is a condition that raises your risk for lung and other diseases.

• The______ is a protein made in your liver to help protect the lungs so if your body does not make enough Alpha One antitrypsin, your lungs are more easily damaged from smoking pollution or dust.

ALPHA-1-ANTITRYPSIN DEFICIENCY

alpha one antitrypsin

How well did you know this?

Not at all

Perfectly

• the patient loses serum albumin and low molecular weight proteins in the urine and some IGG
• At the same time an increase occurs in alpha 2 macroglobulin beta lipoprotein components and the haptoglobin
• These events leads to dramatic decrease in the relative amount of albumin and a significant increase in the relative amount of alpha 2 globulin and beta globulin fraction

Nephrotic syndrome

How well did you know this?

Not at all

Perfectly

result in decrease in albumin and increase in a-1 globulin, a-2 globulin and beta globulin.

Inflammation

How well did you know this?

Not at all

Perfectly

Also called an Acute Phase Reactant pattern

• These patterns are seen in trauma, burns, infarction, malignancy and liver diseases.

Inflammation
| ^
V Albumin | Globulins

How well did you know this?

Not at all

Perfectly

• In infectious _____ the gamma globulin fraction rises with increasing hepatocellular damage

• In obstructive jaundice there is an increase in the a-2 and beta-2 globulins. Also noted in the obstructive jaundice is an increased concentration of lipoprotein which is an indicator of its biliary origin.

CIRRHOSIS

TC N, ↓

A ↓

G ↑

Hepatic damage

Burns, trauma

Infections

B-y bridging

Cirrhosis

• ↑ y-globulins

Hepatitis

• ↑ a2, B-globulins

Obstructive jaundice

Infections • ↑ a1-a2-globulins • ↑ a1-a2-y-globulins

Acute Chronic

TC ↓ A ↓ G N

Malabsorption Inadequate diet Nephrotic syndrome ↑a1-, B-globulins; ↓y-globulins

TC ↓ A N G ↓

Immunodeficiency syndromes

TC ↓ A ↓ G ↓

Salt retention svndrome

TC ↑ A ↑ G ↑

Dehydration

TC ↑ A N G ↑

Multiple myeloma Monoclonal and polyclonal gammopathies

• Modifying electrophoretic parameters (12 bands) • Uses higher voltage coupled with a cooling system and more conc. buffer.

HIGH-RESOLUTION PROTEIN ELECTROPHORESIS

HIGH-RESOLUTION PROTEIN ELECTROPHORESIS •_______- support medium •_____ estimates of protein • useful in detecting____ monoclonal bands and differentiating unusual bands

Agarose gel semiquantitative small

• Separation of molecules takes place in silica capillaries • Capillaries are typically 30-50 cm long

CAPILLARY ELECTROPHORESIS

CAPILLARY ELECTROPHORESIS • Separation of molecules takes place in____ • Capillaries are typically____ long

silica capillaries 30-50 cm long

• Zone electrophoresis that separates proteins on the basis of pl

ISOELECTRIC FOCUSING

• Specific proteins may be identified by immunochemical assays in which the reaction of protein (antigen) and antibody is measured.

IMMUNOCHEMICAL METHODS

IMMUNOCHEMICAL METHODS (6)

Radial immunidiffusion immunoelectrophoresis immunofixation electrophoresis electroimmunodiffusion immunoturbidimetry immunonephelometry

IMMUNOCHEMICAL METHODS Fluorophore Enzyme Gold particles

Marker

IMMUNOCHEMICAL METHODS Antigen

Target protein

Inside a damaged kidney there is a presence of

Albumin

• resulting from glomerular or tubular dysfunction

Proteinuria

URINARY PROTEINS Qualitative test: Quantitative test:

Reagent test strip 12-24 hour urine specimen

URINARY PROTEINS Precipitation methods:

Sulfosalicylic acid (SSA) Trichloroacetic acid (TCA) Benzethonium chloride

URINARY PROTEINS Chemical Methods:

Biuret Method Folin-Lowry Method

URINARY PROTEINS Dye-binding methods:

Coomasie blue Ponceau S

Proteins are precipitated as fine particles, turbidity is measured spectrophotometrically Rapid, easy to use; unequal sensitivity for individual protein

Turbidimetric methods (sulfosali-cylic acid, trichloroacetic acid, or benzethonium chloride)

Proteins are concentrated by precipitation, redissolved in alkali, then reacted with Cu?t; Cu?+ forms colored complex with peptide bonds Accurate

Biuret

Initial biuret reaction; oxidation of tyrosine, tryptophan, and histidine residues by Folin phenol reagent (mixture of phosphotungstic and phosphomolybdic acids): measurement of resultant blue color Very sensitive

Folin-Lowry

Protein binds to dye, causes shift in absorption maximum Limited linearity; unequal sensitivity for individual proteins

Dye binding (Coomassie blue, Ponceau S)

CSF PROTEINS Reference interval (10-40 years old):

15-45 mg/dL

• Abnormal increased of total CSF Proteins:

Bacterial, Viral, and Fungal Meningitis traumatic tap; multiple sclerosis; obstruction; neoplasm; disk herniation; cerebral infraction

Degree of permeability can be evaluated by measuring the CSF albumin comparing it with the serum albumin. (Ref. value CSF albumin-serum albumin ratio: less than_______)

2.7 to 7.3

CSF PROTEINS Increased: Decreased:

damaged blood brain barrier hyperthyroidism, fluid leakage from CNS