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SDS Page

SDS-PAGE is a technique used to separate proteins by size. It involves preparing protein samples using a buffer containing SDS to denature the proteins and impart a negative charge. This is followed by preparation of a resolving gel and stacking gel, loading the samples into the wells, running electrophoresis to separate the proteins based on size, and staining and visualization to view the separated protein bands. Key aspects include the different pH levels of the gels which help concentrate and then separate the proteins, and comparison to a protein marker or ladder to determine protein molecular weights.

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614 views12 pages

SDS Page

SDS-PAGE is a technique used to separate proteins by size. It involves preparing protein samples using a buffer containing SDS to denature the proteins and impart a negative charge. This is followed by preparation of a resolving gel and stacking gel, loading the samples into the wells, running electrophoresis to separate the proteins based on size, and staining and visualization to view the separated protein bands. Key aspects include the different pH levels of the gels which help concentrate and then separate the proteins, and comparison to a protein marker or ladder to determine protein molecular weights.

Uploaded by

Shadia Heyari
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© © All Rights Reserved
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SDS-PAGE

• SDS-PAGE, sodium dodecyl sulfate


polyacrylamide gel electrophoresis, is a technique
widely used in biochemistry, forensics, genetics and
molecular biology to separate proteins according to
their electrophoretic mobility (a function of length of
polypeptide chain or molecular weight as well as
higher order protein folding, posttranslational
modifications and other factors).The SDS gel
electrophoresis of samples having identical charge
per unit mass due to binding of SDS results in
fractionation by size and is probably the world's most
widely used biochemical method.
• In this technique we will assay the protein purity
according to its molecular weight, so we need to
eliminate all the differences between these proteins
except for the composition. So the unique folding and
the overall charge of each protein must be
eliminated.
1st Step: Sample preparation:
we need to prepare our protein sample that was isolated previously from sheep blood,
we will prepare a buffer from the following components:
1. 12.5 ml 1 M Tris pH 6.8
2. 5 ml glycerol: used to stack the proteins sample inside the well.
3. 2.5 ml 20% SDS: an anionic detergent, denatures secondary and non–disulfide–linked tertiary
structures, and applies a negative charge to each protein in proportion to its mass.
4. 5 ml 2-mercaptoethanol: reducing agent used to disrupt disulfide bonds to ensure the protein is
fully denatured before loading on the gel; ensuring the protein runs uniformly.
5. Tiny bit of Bromophenol blue: tracking dye, has low MWT.

* You won’t add this whole amount of


buffer to one sample. You take from this
buffer the same volume as your sample
in the centrifuge tube.
• After addition of sample preparation buffer, proteins may optionally be briefly
heated to near boiling in the presence of a reducing agent, such as dithiothreitol
(DTT) or 2-mercaptoethanol (beta-mercaptoethanol/BME), which further
denatures the proteins by reducing disulfide linkages, thus overcoming some
forms of tertiary protein folding, and breaking up quaternary protein structure.
• Now the sample is ready, so we can start the process of separation, but first we
need to prepare The Gels of the SDS PAGE….
2nd Step: Gels Preparation Resolving gel
10%
Stacking gel
4%

30% Acryl:bis 3.3 ml 1.33 ml


Gel preparation components (29:1)
1.5 M Tris 2.5 ml 1.25 ml
(pH=8.8)
- We will prepare 2 gels, they have small differences but Stacking gel
mainly composed from the same components: (pH 6.8)
• acrylamide-bisacrylamide: synthetic gel, thermo-stable, H2O 4.0 ml 7.2 ml
transparent, strong, relatively chemically inert,
autopolymerization. 10% SDS 100 ml 100 ml

• 10% Ammonium persulfate (APS): free radical initiator 10%Ammoni 100 ml 100 ml
(initiates gel formation) um persulfate
• TEMED: catalyst for APS.
TEMED 10 ml 10 ml

Total volume 10 ml 10 ml
• Here we will prepare 2 kinds of gel:
1- Resolving gel (Running gel): prepared first.
separation occurs here as the level of Bis-acrylamide is
higher here, has basic pH=8.8
2- Stacking gel ( Loading gel): prepared after
solidifiction of the resolving gel. This gel is cast over
the resolving gel. The height of the stacking gel region
is always maintained more than double the height and
the volume of the sample to be applied. here we load
our samples, its pH= 6.8
• Gels are polymerized in a gel caster. First the
separating gel is poured and allowed to polymerize.
Next the loading gel is poured and a Comb is placed
to create the wells (where we add our protein
sample). After the loading gel is polymerized the
comb can be removed and the gel is ready for
electrophoresis.
3rd Step: Electrophoresis
• The electrophoresis apparatus is set up with running buffer
covering the gel in the chamber (to allow conduction of
electricity through the gel systems). Next, the sample is added
to the wells in the gel with a syringe or pipette, we add also
protein marker/ Standard or Ladder that act as a reference, it’s
composed of many proteins together that have different
molecular weights.
• Finally, the apparatus is hooked up to a power source under
appropriate running conditions to separate the protein bands.
• An electric current is applied across the gel, causing the
negatively-charged proteins to migrate across the gel towards
the anode. Depending on their size, each protein will move
differently through the gel matrix: short proteins will more
easily fit through the pores in the gel; while larger ones will
have more difficulty (they encounter more resistance).
• After a set amount of time, the proteins will have differentially migrated based on
their size; smaller proteins will have traveled farther down the gel, while larger ones
will have remained closer to the point of origin. Therefore, proteins may be separated
roughly according to size (and therefore, molecular weight).
• Without SDS, different proteins with similar molecular weights would migrate
differently due to differences in mass/charge ratio, as each protein has an isoelectric
point and molecular weight particular to its primary structure.
• the tracking dye added to the protein solution, allows the experimenter to track the
progress of the protein solution through the gel during the electrophoretic run.
Why is pH important in SDS-PAGE?
• SDS-PAGE is divided into resolving gel (high pH) and
stacking gel (low pH). The stacking gel helps concentrate
the proteins on their way to the resolving gel.
• This is because Glycine molecules in the running buffer
appears as zwitter ions under low pH, leading to electric
field strength between the negatively charged Cl-( Cl- ions
present in the gels) ions and the glycine molecule.
• When entering the separating gel the glycine molecules
appears as –ve charged (high pH) and will outrun your
protein molecules which can be divided to their molecular
sizes.
SDS-PAGE Protein
marker
• Following electrophoresis, the gel may be
stained most commonly with Coomassie
Brilliant Blue (today’s lab) R-250 or silver
stain, allowing visualization of the
separated proteins. After staining, all of
the gel will appear blue in color so we will
apply destaining solution (5-10%
methanol, 10% acetic acid) so that
different proteins will appear as distinct
bands within the gel.
• Then we will compare the size of the
band with the reference protein marker
to know the size of the protein of our
sample.
Please watch the following 2 videos to
undertand everything about SDS PAGE:
https://www.youtube.com/watch?v=i_6y6Z5UvwE&feature=youtu.be&fbclid=IwAR1hH_J1CK
WQmnVfA5ivtn8sg5HTSnfThoCP-hLNPSEWd2rH3zr6BxycZ7Y

https://www.youtube.com/watch?v=eaETFKXtNRA&feature=share&fbclid=IwAR1C-
PxHFbtC12JgqD1I49LIbIdfjeNe7axpTofJf4IaxJvnAUewQx4JsZw

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