Exp 7-SDS-PAGE

EXPERIMENT 7
SDS-PAGE
GEL ELECTROPHORESIS OF
PROTEINS (SDS-PAGE)
 Electrophoresis is the process of moving charged molecules in solution by
applying an electric field across the mixture. Because molecules in an electric
field move with a speed dependent on their charge, shape, and size,
electrophoresis has been extensively developed for molecular separations.
 Sodium Dodecyl Sulfate-Polyacrylamide gel Electrophoresis (SDS-PAGE), is
a technique widely used in biochemistry, forensics, genetics and molecular
biology to separate and identify proteins according to their molecular weight.
 Molecules flow at different rate depends on the molecular size of proteins
SDS-PAGE
 The gel, a matrix of pores which allow the molecules migrate at different rates,
used for SDS-PAGE is made out of acrylamide which form cross-linked
polymers of polyacrylamide.
 The size of pores is determined by the concentration of acrylamide. The higher
the concentration, the smaller the size of pores (denser matrix). Denser
matrices are used for separating smaller proteins; larger proteins may find the
pores in a dense matrix too small to enter, and may therefore not enter the gel
at all. The table below lists approximate useful ranges for different gel
densities
SDS-PAGE
 SDS PAGE allows:
 Assessment of purity of the
preparation
 Estimation of approximate
quantity of the protein, and
 Measurement of the size of the
protein.
SDS-PAGE
 Sodium dodecylsulfate (SDS) is negatively
charged detergent used to denature, linearize the
proteins and coat the proteins with negatively
charged.
 A Concurrent treatment with a disulfide reducing
agent such as β-mercaptoethanol or DTT
(dithiothreitol), which further denatures the
proteins by reducing disulfide linkages, thus
overcoming some forms of tertiary protein folding,
and breaking up quaternary protein structure. So,
the proteins samples are having uniformed structure
and charge and the separation will depend on their
molecular weight only.
SDS-PAGE
 Standard gels are typically composed of two layers:
 The stacking layer (top gel) contains a low percentage of
acrylamide (6%) and has low pH (6.8)
 Separating gel (bottom gel) acrylamide concentration of
the separating gel varies according to the samples to be run
(5%-15%) and (10% for your experiment) and it has
higher pH (8.8).
 The difference in pH and acrylamide concentration at the
stacking and separating gel provides better resolution and
sharper bands in the separating gel.
SDS-PAGE PREPARATION:
 Sample Preparation:
 protein sample + sample buffer (boil the mixture in heating block at 95oC for 5
min)
 5x sample buffer ( Protein Loading Dye) contain:
 2% (w/v) SDS
 60mM Tris/HCl, pH 6.8
 25%Glycerol
 14.4 mM β-Mercaptoethanol
 1% Bromophenol blue
 The tracking dye makes it easier to see the sample during loading, and lets you know when the
lowest molecular weight proteins are getting near the end of the gel.
 The sample buffer contains glycerol to make the protein sample denser than the
electrophoresis tank buffer, so that the protein sample will sink to the bottom of the well when
you load it.
 Acrylamide stock should be prepared first:
 Cross-linked polyacrylamide gels are formed from the polymerization of
acrylamide monomer in the presence of smaller amounts of N,N’-methylene
bisacrylamide (normally referred to as ‘bis’-acrylamide) which helps in
crosslinking the polymers.
 10% resolving gel preparation:
 1.5 ml 1.5 M Tris-HCl, pH 8.8
 2 ml 30% acrylamide/Bisacrylamide
 0.06 ml 10% SDS
 2.44 ml water
 Add last to initiate reaction:

 30 μl 10 % ammonium persulfate (initiate polymerization) and 5 μl TEMED
(accelerate polymerization)
 Pour gel and layer n-butanol or isopropanol on top of the polymerizing solution.
After polymerization is complete, rinse off the top of the gel to remove the
butanol.
 Stacking gel preparation:
 0.875 ml 1.0 M Tris-HCl, pH 6.8
 0.583 ml 30% acrylamide/Bisacrylamide
 0.035 ml 10% SDS
 2.007 ml water
 Add last to initiate reaction:

 30 μl 10 % ammonium persulfate and 5 μl TEMED
 Pour the gel, insert the comb to create the wells and leave to congeal.
 Loading sample and Running the gel using, (Tris Glycine Buffer)
 Running buffer (tank buffer) contains:
 25 mM Tris
 192 mM glycine, pH 8.8
 0.1% SDS
 Add the tank buffer, remove the comb to produce wells and load
the samples
 The molecular weight standards (marker) can be used to
calibrate the migration of proteins of differing sizes on the gel. For
any given gel, the migration will be inversely proportional to the
log of the molecular weight.
 Stain the gel using staining buffer, Coomassie Staining
Solution (10% Acetic acid, 25% Methanol, 0.05% Coomassie
R-250 or Bio-Safe Coomassie staining solution)
 Coomassie blue is a dye that binds proteins.
 The function of the acetic acid and methanol is to cross-link
the proteins into the gel so that they do not diffuse.
 De-stain the gel using De-staining buffer, It contains:
 Glacial acetic acid
 Methanol
 Which results in removal of the excess Coomassie blue.

 See colored bands under white lamp light
PROCEDURE
 1. Set up the gel apparatus.
Mark location of wells before adding tank buffer, Remove bubbles from bottom of
gel. The gel apparatuses you will be using have 10 wells, so you can plan on running
3 lanes for each of your samples. Each sample should contain 20 μl to which you
will add 5 μl 5x sample buffer. Prepare 2 μg, 10 μg, and 50 μg aliquots for the crude
homogenate and desalted ammonium sulfate samples; prepare 2 μg and 10 μg
aliquots for the peak fraction(s). (Note: do not prepare the 50 μg sample for your
peak fraction(s); this would result in an overloaded lane.) To calculate how much of
each sample to use you must refer back to the protein concentration determined by
the Bradford assay in Experiment 6. If your protein concentration is too low to allow
preparation of the more concentrated aliquots, make the most concentrated aliquots
you can.
PROCEDURE
 2. Add 5x sample buffer to each sample. The sample buffer should be added at 1/5 the final
volume. Your samples contain 20 μl so you should add 5 μl sample buffer (5μl is 1/5 of 25
μl). If your samples do not contain 20 μl of diluted protein, you must determine how much
sample buffer to add.
 3. Calculate how you are going to prepare all of your samples before coming to class!
 4. Heat all samples at 100°C for 2 minutes. Spin down the protein solution for 5 seconds, so
that all of the liquid is at the bottom of the microfuge tube.
 5. Apply samples to wells. Record how the samples were loaded!
 6. Attach the electrodes to gel apparatus, and turn on the power supply. Run the gel at
constant current (15 mA) until the tracking dye reaches 1 cm from the end of the gel. Be
careful-the high current during gel electrophoresis is dangerous!
PROCEDURE
 7. Turn off the power supply and remove electrodes. Remove the gel sandwich.
 Remove one spacer, and insert it in one corner between the glass plates. Use the spacer to
gently pry off one glass plate. Carefully remove the gel from the plate and cut one lower
corner at lane #1 to distinguish the two sides. Place it in a weigh boat containing stain
solution.
 Do not handle the gel with your bare hands: acrylamide is a cumulative neurotoxic agent and
is absorbed through the skin! Residual unpolymerized acrylamide will be present in the gel.
 pre-cast gels are used because they are convenient and reproducible. Pre-cast gels are also
desirable because they preclude exposure to the relatively toxic monomers used in gel
production
 The gel will be stained overnight and transferred to the destaining solution before the next lab
period.
SUMMARY
 SDS-PAGE
 https://www.youtube.com/watch?v=-H8GU_90Gak

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EXPERIMENT 7

 Add last to initiate reaction:

 Add last to initiate reaction:

 Which results in removal of the excess Coomassie blue.

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