Student …………………………………………….. Group ……….……….. Data ……………….…
SERUM PROTEIN AND LIPOPROTEIN ELECTROPHORESIS
IN AGAROSE GEL
I.
Serum protein electrophoresis is used to identify the presence of abnormal proteins, to identify the absence
of normal proteins, and to determine when different groups of proteins are present in unusually high or low
amounts in blood or other body fluids.
Proteins in gel are made visible by staining them with dyes
1. Electrophoresis conditions (fill in the gaps):
a. gel type and its concentration: _______________
b. buffer pH = ____________
c. overall charge of proteins: _____________
d. voltage: ______________;
e. separation time: __________________;
f. colour marker to monitor the process: ________________
2. Scheme of applying samples:
Mark the electrophoresis direction, name the electrodes and define their charges.
3. Post-electrophoresis treatment of plate
a. fixation; write down the composition of a fixative solution:
___________________________________________________________________________
b. pressing, washing – write down the name and concentration of a washing solution:
___________________________________________________________________________
c. pressing and drying
ELECTROPHORESIS 1
4. Gel staining – name a dye used in the staining:
a. specific – for all the serum proteins: ___________________________
b. non-specific – for only lipoproteins: ___________________________
5. Destaining of background - by washing with destaining solution (write down its composition):
__________________________________________________________________________________
6. Evaluation of a protein fraction
Electropherogram – a visualisation of human serum proteins electrophoresis;
Densitogram – a diagram obtained as a result of protein fractions’ optical density evaluation using
a densitometer.
Mark protein fractions on both, electropherogram and densitogram.
Mark the electrophoresis direction, name the electrodes and define their charges.
Write down the normal percentage ranges for each protein fraction:
The normal range of total protein in human serum is
_____________________ g/dL (grams per deciliter)
or _____________________ g/L
Because disease states affect the relative amounts of albumin and globulin, the A/G ratio may provide a clue
as to the cause of the change in protein levels
The normal albumin/globulin ratio is _______________________
ELECTROPHORESIS 2
II.
Below you can see the result of normal serum lipoproteins electrophoresis: electropherogram on the left
and densitogram on the right.
Mark lipoprotein fractions on both, electropherogram and densitogram.
Mark the electrophoresis direction, name the electrodes and define their charges.
Write down the normal percentage ranges for each lipoprotein fraction:
Total cholesterol in your blood includes both low-density lipoprotein (LDL) cholesterol and high-density
lipoprotein (HDL) cholesterol.
The acceptable total cholesterol concentration in your blood should be less than ________________ mg/dL
The borderline of total cholesterol concentration for adults is: _______________________________ mg/dL
Other tasks:
1. Explain the term of ‘electrophoresis’ and factors responsible for relative mobility of macromolecule in
porous medium?
ELECTROPHORESIS 3
2. Tick sentences - true about electrophoresis:
a. The pore size in the polyacrylamide gel can be controlled during its polymerisation phase.
b. PAGE (Polyacrylamide gel electrophoresis) can separate macroions in native as well as in reductive
conditions.
c. SDS is an anionic detergent which denatures proteins and gives them an overall positive charge.
d. SDS-PAGE method can separate macromolecules according to their sizes.
e. Electrophoresis in the presence of SDS - the fastest are the biggest macromolecules.
f. Electrophoresis in the presence of SDS - the fastest are macromolecules with the greatest charges.
3. Serum protein and lipoproteins electrophoreses are routinely carried out at pH 8.6 and 1% agarose.
Tick the correct sentences:
a. The main serum proteins are albumins; their band is homogenic and the most intensively stained.
b. Gamma-globulins move the fastest towards anode (in comparison to other serum proteins).
c. Chronic inflammation response generates an increase in gamma-globulin region which is also more
intensively stained as compared to the physiological state.
d. Electrophoretic mobility of lipoproteins depends on the presence of apolipoproteins.
4. The addition of β-mercaptoethanol (reductor) affects macromolecules during electrophoresis, because
proteins (mark true sentence/s):
a.
b.
c.
d.
e.
f.
form dipolar ions (zwitterions).
move towards positive electrode.
precipitate out of solution.
reveal the subunit structure.
lose their tertiary structure.
all have the same charge densities.
5. Serum proteins were separated by agarose gel electrophoresis (1% gel, pH 8.6) and stained with Amido
Black.
Draw the protein fractions possibly visible after staining. Write down their names.
Mark the electrophoresis direction, name the electrodes and define their charges.
6. 500 mg of lyophilised protein was dissolved in a buffer to the final volume of 25 cm3 and concentration
of 0,0001 mole/dm3, and density of 1 g/cm3. Calculate the molecular weight of the protein.
ELECTROPHORESIS 4