PROTOCOLS-SUMMER SEMESTER
ISOLATION, DETECTION AND SEPARATION OF PROTEINS – PART 2
Separation of proteins by SDS-PAGE gel electrophoresis
A) Gel cassette preparation
1)
Take a spacer plate and a short plate. Clean them by ethanol and let them dry. Prepare
the plates for gel pouring according to Fig. 1-4 of the Assembly Guide Protocol (see part
Gel Cassette Preparation)
2)
Pour into the space between the plates small amount of ethanol to check the right
position of the plates. If there is no leaking, remove ethanol.
After this leakage test the gel cassette is ready for pouring the gel.
3)
Place a comb completely into the assembled gel cassette. Mark the glass plate 1 cm
below the comb teeth. This is the level to which the resolving 10% gel is poured.
Remove the comb.
4)
Take the tube with premixed 10% separating gel and add 2.5 l TEMED and 25 l 10%
APS. Mix by inverting the tube several times.
By adding APS and TEMED polymerization of the gel is started. From this moment it is
important to work quickly to avoid polymerization of the gel before pouring between
plates.
5)
Pour the solution smoothly to the mark using a pipette with the blue tip or disposable
plastic pipette. Avoid forming bubbles.
6)
Immediately overlay the monomer solution with ethanol.
Ethanol defends the contact of polymerizating gel with oxygen in the air that inhibits the
polymerization process. Ethanol also removes bubbles from the surface of the gel and
helps to form straight horizontal level of the gel.
7)
Allow the gel to polymerize for 30-45 minutes.
When the rest of the gel in the tube gets a gel structure, you can start with the stacking
gel preparation.
8)
After complete polymerization of the gel, remove ethanol and dry the top of the
resolving gel with filter paper.
9)
Take the tube with 4% premixed stacking gel. Add 2 l TEMED and 10 l 10% APS.
Mix by inverting the tube several times.
10) Pour the stacking gel to the space between plates until the top of the short plate is
reached.
11) Insert the comb between the spacers. Avoid making any air bubbles.
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PROTOCOLS-SUMMER SEMESTER
ISOLATION, DETECTION AND SEPARATION OF PROTEINS – PART 2
Bubbles affect the electric conductivity of the gel and so they would affect the separation
process.
12) Allow the stacking gel to polymerize for 30 – 45 minutes.
Complete polymerization of the gel can be expected when the rest of the gel in the tube
has a gel structure.
For separation we use 2 gels with different concentration of acrylamide and different
pH. The stacking gel enables proteins to reach the interface between both gels at the
same time, so that the zone of proteins loaded to the wells became sharp. The mobility
in the separating gel is then dependent only on the size of proteins separated and it is
not affected by other factors.
13) Leave the gel you have just made to polymerize and continue working with the gel,
which was prepared for you by previous study group. The gel you have prepared will be
used by next study group.
B) Preparation of samples
1)
Sign three new 1.5 ml tubes: write letter of your working group and type of the tissue.
2)
Thaw the sample you prepared last week. Pipette 30 l of each protein sample into a
new 1.5 ml tube.
3)
Add 30 l of sample buffer to each protein sample. Mix well.
The sample buffer contains SDS, a detergent, that denaturates proteins and give them
negative charge.
4)
Heat your lysate samples diluted in sample buffer at 95°C for 4 minutes in a heated
thermo block.
The SDS-PAGE System is a discontinuous buffer system that incorporates SDS in the
buffer. In this system, proteins are denatured by heating them in sample buffer
containing sodium dodecyl sulfate (SDS) and a thiol-reducing agent such as 2mercaptoethanol. The resultant polypeptides take on a rod-like shape and a uniform
charge-to-mass ratio proportional to their molecular weights. Proteins separate only
according to their molecular weight, making the system extremely useful for calculating
molecular weights.
C) Loading of samples
1)
Calculate the volume of each sample needed when you want to load 50 g of protein per
well.
For calculation, use concentration of your protein samples that you determined last
week with the Bradford method.
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PROTOCOLS-SUMMER SEMESTER
ISOLATION, DETECTION AND SEPARATION OF PROTEINS – PART 2
2)
Prepare the electrophoresis unit for the loading of the samples according to Fig. 1-5 of
the Assembly Guide (see Part Electrophoresis Module Assembly).
3)
After electrophoresis unit is assembled fill the inner chamber with running buffer until
the level reaches halfway between the tops of the taller and shorter glass plates of the gel
cassettes.
If the level of buffer is lower than the top of the shorter glass, the electric power can not
run threw the gel and the separation do not works.
4)
Add running buffer to the lower buffer chamber.
5)
Load protein markers and actin + myosin sample according to instructions given you by
the head of the practice.
SDS PAGE Protein markers are used for precise molecular weight determination. They
also allow monitoring of separation of proteins in the gel, because they are prestained
with covalently attached dyes. The molecular weights we use are 10, 15, 20, 25, 37, 50,
75, 100, 150 and 250 kDa.
Load all samples slowly to allow them to settle evenly on the bottom of the well.
6)
Load your samples – load doubled volume as the volume you calculated for 50 g of
protein per well.
You have diluted your sample 1:1 with sample buffer, so it is neccesary to multiply the
volume you calculated by 2 when you want to load 50 g of protein per well.
D) Gel electroforesis
1)
Place the lid on the electrophoresis unit. Make sure to align the color coded banana
plugs and jacks. Insert the electrical leads into a suitable power supply with the proper
polarity.
2)
Begin electrophoresis: 200 V constant. Run time is approximately 35 minutes.
The bromphenol blue which is a part of the loading dye has the highest electric mobility
of all components of the samples loaded on the gel. When it reaches the end of the gel
electrophoresis should be stopped to avoid release of proteins to the running buffer.
3)
After electrophoresis is complete, turn off the power supply and disconnect the electrical
leads. Take the glasses with the gel and separate the gel from the glasses.
E) Staining of the gel and identification of actin
1)
Put the gel carefully into Coomassie blue staining solution. Stain with mild agitation
approximately for 10 minutes.
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PROTOCOLS-SUMMER SEMESTER
ISOLATION, DETECTION AND SEPARATION OF PROTEINS – PART 2
Coomassie Blue is a protein binding substance that enables visualization of proteins.
2)
Subsequently put the stained gel into Coomassie blue destain solution to destain the
background of the gel. Then continue in destaining with hot water.
During destaining the background of the gel particular bands of proteins become
visualized.
3)
According to molecular weight markers and actin and myosin samples identify actin and
myosin in the separated proteins of your samples. Compare the amount of actin and
myosin among the different types of tissue
- Actin and myosin are highly conservative proteins. Their size and structure do not
differ very much among different species. The molecular weight of actin is
approximately 43 kDa. Myosin consists of heavy chain with molecular weight of 210
kDa and several light chains of 15-20 kDa.
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