final sds ppt.pptx

Presentors
Areej Ahmed & Marryam Faiz
Z19-24 & Z18-15

Jujges
Muhammad Ibrahim Miraal Shehzad Hafsa Malhan

Contents:
History
Introduction
Components and apparatus
Principle
Procedure
Applications
Advantages and disadvantages

History
• In 1948, Arne Tiselius was awarded the Nobel Prize in Chemistry for the discovery of the
principle of electrophoresis .
• The discontinuous electrophoresis of 1964 by L. Ornstein and B. J. Davis made it possible to
improve the separation by the stacking effect.
• The denaturing effect of SDS in continuous polyacrylamide gels was first described in 1965
by David F. Summers in the working group of James E. Darnell to separate poliovirus
proteins

….
• The SDS-PAGE was described in 1970 by Ulrich K.
• But the technique was actually invented by Jake Maizel, who was doing a sabbatical in the
MRC laboratory when Laemmli joined the lab as a postdoctoral fellow.
• Maizel shared his prior technology with Laemmli and together they made further
improvements. Laemmli and Maizel had planned to follow up with a Methods paper but this
never materialized.

Introduction
SDS PAGE is a technique used for the separation of proteins based on their molecular weight.
SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)
SDS-PAGE is an electrophoresis method that allows protein separation by mass.
Principle:
I. The principle of SDS-PAGE states that a charged molecule migrates to the electrode with the
opposite sign when placed in an electric field.
II. The separation of the charged molecules depends upon the relative mobility of charged
species.
III. The smaller molecules migrate faster due to less resistance during electrophoresis.

Material required 1. Power supplies.
2. Gels.
3. Electrophoresis chambers.
4. Protein samples.
5. Running buffer.
6. Loading buffer.
7. Staining and destaining buffer.
8. Protein ladder.
9. Reagents

SDS
1. sodium dodecyl sulfate (SDS) is a popular anionic detergent for routine protein
electrophoresis and cell lysis methods.
2. SDS is also present in the gel to make sure that once the proteins are linearized
and their charges masked, they stay that way throughout the run.
3. This product is a powder ,for preparing SDS solution, dissolve the powder in H2O.

….
How to prepare SDS solution:
1. Weigh out 10 g SDS and add to a 100 mL Duran bottle. Be careful ,avoid inhalation and use a fu
me mask.
2. Measure out 80 mL of distilled water and add to the Duran bottle.
3. Place on a magnetic stirring plate to mix the solution.
4. To dissolve SDS quicker, you can gently heat up the solution (60oC) using a hot plate or water ba
th.
5. Once the powder has fully dissolved, top up the solution to 100 mL using distilled water.
6. An optional step is to filter the solution to remove any particles that have failed to dissolve.

….
1. The function of SDS is to break the
hydrogen bonds of proteins
2. Masks the charge on protein so that
all protein will act as negatively charged.
3. so that separation of the proteins is solel
y based on molecular weight.
4. It is a denaturing reagent.

Beta mercaptoethanol
1. Beta-mercaptoethanol (BME) is a red
ucing agent that acts on disulfide bonds.
2. In the absence of BME, proteins with
disulfide bonds retain some of their
structure
3. BME
4. BME is a poison if ingested, causes irrit
ation in the mucous membranes, and can
be absorbed dermally.

PAGE
Polyacrylamide gel electrophoresis.
Principle: when a charged molecule is exposed to an electric field, it migrates towards the electrode
carrying the opposite charge.
Function: PAGE is a biochemical technique that allows for proteins to be separated by
their electrophoretic mobility .
The gel is prepared by polymerizing acrylamide with the cross-linking agent
N,N′-methylenebisacrylamide (bis-acrylamide) with the addition of ammonium persulfate .

….
Polyacrylamide:
PAM is highly water-absorbent, forming a soft gel when hydrated
Acrylamide : a colorless crystalline solid which readily forms water-soluble polymers. They form
linear polymers
Bisacrylamide :acts as the cross-linking agent, creating a network of polyacrylamide instead of
linear polymers.
The 'pore size' is determined by the ratio of acrylamide to Bisacrylamide.
Ammonium persulfate: Catalyses the polymerization of acrylamides and poly acrylamides.
TEMED: is used with ammonium persulfate (APS) to catalyze acrylamide polymerization when pr
eparing gels for electrophoresis.
Thermo Scientific Pierce Tetramethylethylenediamine.

Gel
In SDS-PAGE, the gel is cast in a buffer containing sodium dodecyl sulfate (SDS), an anionic detergent.
1. Stacking gel:
The purpose of the stacking gel is to concentrate all of the different sized proteins into a compact
horizontal zone by sandwiching them between a gradient of glycine molecules above and chloride ions be
ow.
It has lower amount of polyacrylamides.
2. Running gel:
Based on their size and charge, the molecules will travel through the gel in different directions or
at different speeds, allowing them to be separated from one another.
It has higher amount of polyacrylamide.

Electrophoresis:
Electro: charge
Phorsesis :carry
“It is the movement of charged particles in a fluid or gel under the influence of an electric field.”

Loading buffer
Loading buffers are reagents added to protein or DNA samples for loading into gels for electrophoresis
applications. Loading buffers contain a colored dye, most commonly blue or orange, to visualize and track
the progression of the bands across the gel.
It contains:
1. 10% SDS
2. B-mercaptoethanol 10%
3. 20 % Glycerol
4. 0.12M Tris-HCL
5. 0.004% bromophenol blue dye
6. Sample

Running buffer
The running buffer contains ions that conduct current through the gel.
Tris-HCl 25 mM
Glycine 200 mM
SDS 0.1% (w/v)

Staining and destaining
Once proteins are separated by gel electrophoresis, staining can be used to visualize the proteins.
The most common method is staining with Coomassie blue, which after washing gives blue bands on a clear bac
kground.
Destain Solution is a uniquely formulated, ready-to-use reagent specifically designed for in-gel visualization of
Coomassie Blue stained total protein in polyacrylamide gels.
Continue the destaining until the protein bands are seen without background staining of the gel.
Destain the gel by soaking for at least 2 hours
10% acetic acid.
50% methanol.
40% H2O .
If the gel still has a Coomassie Blue background then continue destaining until the background is nearly clear

Apparatus
Voltage :200v
Current:100mA to 120 mA

• https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/agaro
se-gel-electrophoresis#:~:text=Proteins%20are%20generally%20smaller%20than,(sieve)
%20size%20than%20agarose.
• https://www.researchgate.net/post/Why-gel-electrophoresis-of-protein-is-vertical-while-g
el-electrophoresis-of-DNA-is-horizontal
• https://www.protocols.io/view/assessing-protein-purity-using-sds-page-cegktbuw.html#:~
:text=SDS%2DPAGE%20allows%20an%20estimation,the%20standard%20or%20protein
%20ladder.
• https://www.researchgate.net/post/Why_do_we_run_SDS_PAGE_in_vartical_position
• https://www.lonzabio.jp/catalog/pdf/pd/PD014.pdf
• https://pubmed.ncbi.nlm.nih.gov/22585481/
• https://en.wikipedia.org/wiki/SDS-PAGE#Analysis
References

Why Agarose is not used instead of polyacrylamide?
• Proteins are generally smaller than DNA.
• Thus, while DNA (larger than 100 bp) is routinely separated on agarose gels, proteins
are generally run on polyacrylamide gels, as polyacrylamide matrices have a smaller
pore (sieve) size than agarose.
• Polyacrylamide is inert.

Principle
• A charged molecule migrates to the electrode with the opposite sign when placed in an electric
field. The separation of the charged molecules depends upon the relative mobility of charged
species.
V=qE/f
• The smaller molecules migrate faster due to less
resistance during electrophoresis.
• Sodium dodecyl sulphate and polyacrylamide
eliminate the influence of structure and charge
of the proteins, and the proteins are separated
based on the length of the polypeptide chain.

Procedure
Gel Preparation
01
Sample Preparation
02
Electrophoresis
03
Gel Staining
04
Destaining
05

1- Gel Preparation
1. Clean the glass plates and spacers of the gel casting unit with deionized water and
ethanol.
2. Assemble the plates with the spacers on a stable, even surface.
3. Two types of gel are prepared
• Separating gel
• Stacking gel

1. Prepare resolving gel solution using the following volumes (for 10 mL) depending on the
percentage of gel required.
Gel % Water (mL)
30% acrylami
de (mL)
1.5 M Tris-HCl
, pH 8.8 (mL)
10% SDS (µ
L)
10% APS (µ
L)
TEMED*(µL)
8% 4.6 2.6 2.6 100 100 10
10% 3.8 3.4 2.6 100 100 10
12% 3.2 4.0 2.6 100 100 10
15% 2.2 5.0 2.6 100 100 10
*TEMED must be the last ingredient added
1- Separating Gel
2. Pour the gel solution in the plates assembled with spacers. To maintain an even and horizontal
resolving gel surface, overlay the surface with water or isopropanol
3. Allow the gel to set for about 20-30 min at room temperature.

1- Separating Gel
1. Pour acrylamide solution for a separating gel.
2. Overlay with water to prevent contact with air (oxygen), which inhibits polymerization.
3. Allow acrylamide to polymerize for 20-30 minutes to form a gel. Remove the overlaid water.

• Proteins migrate at different rate depending on the concentration of the separating gel.
• Use an appropriate gel concentration for your target protein.
• Using a higher acrylamide concentration produces a gel with a smaller mesh size suitable
for the separation of small proteins. In general, an acrylamide concentration between 6 a
nd 15% is used.
• Gels with an acrylamide concentration gradient (gradient gels) are also used.

1. Prepare stacking gel solution.
2- Stacking Gel
Gel %
Water (
mL)
30% ac
rylamid
e (mL)
0.5 M T
ris-HCl,
pH 6.8 (
mL)
10% S
DS (µL)
10% AP
S (µL)
TEMED
*(µL)
5%
5.86 1.34
2.6 100 100 10
*TEMED must be the last ingredient added

1. Pour acrylamide solution for a stacking gel until it overflows.
2. Insert a comb and allow the acrylamide to polymerize ensuring no air bubbles
are trapped in the gel or near the wells.
3. Allow the gel to set for about 20-30 min at room temperature.
2- Stacking Gel

• Proteins are highly concentrated when they
migrate through a stacking gel prior to
entering a separating gel.
• The concentration occurs due to the
difference in the migration rate of glycine
ion, chloride ion, and proteins.
2- Stacking Gel

2- Sample Preparation
1. Add sample buffer to samples (equal volume) and mix by flicking the tube.
2. Heat the samples at 100°C for 3 minutes in a heat block.
3. Centrifuge at 15,000 rpm for 1 minute at 4°C, and use the supernatant for SDS-PAGE.

3- Electrophoresis
1. Remove the binder clips, spacer, and comb from the gel assembly, and mount the gel in
the electrophoresis apparatus using binder clips.
2. Pour running buffer into the upper and lower chambers of the electrophoresis apparatus
and remove air bubbles and small pieces of gel from the wells and under the gel using a
syringe.

3-Electrophoresis
3. Load samples and molecular weight markers in wells.

3-Electrophoresis
4. Turn on the power supply, and run the gel until the dye (BPB) in the sample buffer reaches
the bottom of the gel.

3-Electrophoresis
5. Remove the gel assembly from the electrophoresis apparatus. Remove the gel from the
glass plates using a spatula, and prepare for subsequent analysis.

4- Gel Staining
1. Coomassie Blue staining: Staining of protein gels with Coomassie Brilliant Blue
is a common procedure to visualize proteins resolved by SDS-PAGE.
2. Prepare Coomassie blue solution
Coomassie Stain Solution: (1 l)
Coomassie Blue 2 g
Methanol 450 ml
dH2O 450 ml
Glacial acetic acid 100 ml
3. Place resolving gel in Coomassie Stain with gentle agitation for 30 min (note: the
gel will shrink).

5- Destaining
1. Destaining solution is prepared.
Methanol 50 ml
Glacial acetic acid 70 ml
dH2O …to 1 l
2. The gel is soaked in the destaining solution for
atleast 2 hours.
3. Destain may need replacing several times. To speed
up destain, place a tissue in the tray with gel to soak
up coomassie.

Why the apparatus is kept vertically instead of horizontally like
that in agarose gel electrophoresis?

Why is the apparatus kept vertically instead of horizontally like
that in agarose gel electrophoresis?
• They comprise a stacking gel and a resolving gel. A vertical arrangement allows you to
make them sequentially. You pour the resolving gel first, and then once it is set, pour the
stacking gel on top of it. This results in one contiguous gel.
• The oxygen in air reacts with free radicals and inhibits the polymerization reaction.
Therefore, this reaction would not proceed efficiently in an open, horizontal system.
• Polyacrylamide gel is very thin compared to agarose gel. In horizontal position you coul
d only load tiny amounts of the sample whereas at least 5 ml is required in each well.

Applications
1. It is used to measure the molecular weight
of the molecules.
2. It is used to estimate the size of the protein
3. Used in peptide mapping
4. Estimate the purity of proteins.
5. Used in HIV test to separate HIV proteins.
6. To analyze post-translational modifications

Advantages and Disadvantages
Advantages
• Cross-linked gel is chemically stable.
• Seperation can be done solely on size
and weight.
• Inexpensive
• Good reproducibility
Disadvantages
• Polyacrylamide and SDS are both toxic and
hazardous, they must be handled carefully.
• Gel preparation is difficult and requires
longer time.
• Deliberate denaturation of proteins prior
to electrophoresis (enzymatic activity, protein
binding interactions generally cannot be
determined by SDS-PAGE)

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