Proteins_ SERUM PROTEIN ELECTROPHORESIS Flashcards
SERUM PROTEIN ELECTROPHORESIS
• Serum are applied close to the_____ end of a support medium (ph_____)
cathode (pH 8.6)
• All major serum proteins carry a net____ charge at ph 8.6 and migrate toward the____.
negative
anode
SERUM PROTEIN ELECTROPHORESIS
5 bands:
Albumin
Alpha-1 globulins
Alpha-2 globulins
Beta-globulins
Gamma-globulins
: travels farthest to the anode
Albumin
= number of proteins present in that fraction.
Width of the band
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
Dyes used in serum protein electrophoresis
Ponceau S
Amido black
Coomassie blue
• 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
• 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
Albumin
Percentage and Concentration
53 - 65%
3.5 - 5.0 g/dL
a-1 globulin
Percentage and Concentration
2.5 - 5%
0.1 - 0.3 g/dL
a-2 globulins
Percentage and Concentration
7 - 13%
0.6 - 1.0 g/dL
Beta-globulin
Percentage and Concentration
8 - 14%
0.7 - 1.1 g/dL
Beta-globulin
Percentage and Concentration
8 - 14%
0.7 - 1.1 g/dL
Gamma globulin
Percentage and Concentration
12 - 22%
0.8 - 1.6 g/dL
” gamma spike”
MONOCLONAL IMMUNOGLOBULIN DISEASE
MONOCLONAL IMMUNOGLOBULIN DISEASE
Gamma spike
_________ 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
• 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
• 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
result in decrease in albumin and increase in a-1 globulin, a-2 globulin and beta globulin.
Inflammation
Also called an Acute Phase Reactant pattern
• These patterns are seen in trauma, burns, infarction, malignancy and liver diseases.
Inflammation
| ^
V Albumin | Globulins
• 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
