International Journal of Science and Research (IJSR)

ISSN (Online): 2319-7064

Impact Factor (2012): 3.358

A Practical Approach on SDS PAGE for Separation

of Protein
Suvra Roy, Vikash Kumar*

Central Inland Fisheries Research Institute (CIFRI), Barrackpore, 700120, India

Abstract: Polyacrylamide gel electrophoresis (PAGE), describes a technique widely used in biochemistry, forensics, genetics, molecular
biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic
mobility. Mobility is a function of the length, conformation and charge of the molecule. As with all forms of gel electrophoresis,
molecules may be run in their native state, preserving the molecules' higher-order structure or a chemical denaturant may be added to
remove this structure and turn the molecule into an unstructured linear chain whose mobility depends only on its length and mass-to-
charge ratio. For nucleic acids, urea is the most commonly used denaturant. For proteins, sodium dodecyl sulfate (SDS) is an anionic
detergent applied to protein sample to linearize proteins and to impart a negative charge to linearized proteins. This procedure is called
SDS-PAGE. In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby
resulting in a fractionation by approximate size during electrophoresis. Proteins that have a greater hydrophobic content, for instance
many membrane proteins, and those that interact with surfactants in their native environment, are intrinsically harder to treat accurately
using this method, due to the greater variability in the ratio of bound SDS.

Keywords: SDS-PAGE, molecular biology, biotechnology, electrophoretic mobility

1. Introduction proteins will be covered with many negative charges. So a

protein that started out like the one shown in the top part of
Sodium dodecyl sulfate polyacrylamide gel electrophoresis figure 1 will be converted into the one shown in the
(SDS-PAGE) is a technique for separating proteins based bottom part of figure 1. The end result has two important
on their ability to move within an electrical current, which features: 1) all proteins retain only their primary structure
is a function of the length of their polypeptide chains or of and 2) all proteins have a large negative charge which
their molecular weight. This is achieved by adding SDS means they will all migrate towards the positive pole when
detergent to remove secondary and tertiary protein placed in an electric field.
structures and to maintain the proteins as polypeptide
chains. The SDS coats the proteins, mostly proportional to
their molecular weight, and confers the same negative
electrical charge across all proteins in the sample.
Glycosylated proteins may not migrate at their expected
molecular weight since their migration is based more on
the mass of their polypeptide chains, not the sugars that are
attached [1]. The most widely used gel system for
separating a broad range of proteins by SDS-PAGE is the
Laemmli system [2] which uses tris-glycine gels
comprised of a stacking gel component (which is used to
help focus the proteins into sharp bands at the beginning of
the electrophoretic run) and the resolving gel where
varying acrylamide gel percentages are used to separate
the proteins based on their mass weight. This classic
system uses a discontinuous buffer system where the pH Figure 1: This cartoon depicts what happens to a protein
and ionic strength of the buffer used for running the gel (pink line) when it is incubated with the denaturing
(Tris pH 8.3) is different from the buffers used in the detergent SDS.
stacking gel (Tris, pH 6.8) and resolving gel (Tris, pH 8.8).
The top portion of the figure shows a protein with negative
2. SDS and positive charges due to the charged R-groups in the
protein. The large H represents hydrophobic domains
Sodium dodecyl sulfate (SDS or NaDS), sodium where nonpolar R-groups have collected in an attempt to
laurilsulfate or sodium lauryl sulfate (SLS) is an organic get away from the polar water that surrounds the protein.
compound with the formula CH3(CH2)11OSO3Na. It is an The lower diagram shows that SDS can disrupt
anionic surfactant used in many cleaning and hygiene hydrophobic areas and coat proteins with many negative
products. SDS is a detergent (soap) that can dissolve charges which overwhelm any positive charges the protein
hydrophobic molecules but also has a negative charge had due to positively charged R-groups. The resulting
(sulfate) attached to it. Therefore, if a cell is incubated protein has been denatured by SDS (reduced to its primary
with SDS, the membranes will be dissolved, all the structure) and as a result has been linearized.
proteins will be solubilized by the detergent, plus all the
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 International Journal of Science and Research (IJSR)
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Impact Factor (2012): 3.358
3. PAGE have access to more of the paths in the polyacrylamide
than of bigger size proteins molecules.
If the proteins are denatured and put into an electric field,
they will all move towards the positive pole at the same
rate, with no separation by size. So we need to put the
proteins into an environment that will allow different sized
proteins to move at different rates. The environment of
choice is polyacrylamide, which is a polymer of
acrylamide monomers. When this polymer is formed, it
turns into a gel and we will use electricity to pull the
proteins through the gel so the entire process is called
polyacrylamide gel electrophoresis (PAGE). A
polyacrylamide gel is not solid but is made of a laberynth
of tunnels through a meshwork of fibers (figure 2 and
figure 3).

Figure 4: Cartoon showing a mixture of denatured

proteins (pink lines of different lengths) beginning their
journey through a polyacrylamide gel (grey slab with
tunnels).

An electric filed is established with the positive pole (red

plus) at the far end and the negative pole (black minus) at
the closer end. Since all the proteins have strong negative
charges, they will all move in the direction the arrow is
pointing (run to red).

Polyacrylamide gel electrophoresis is undoubtedly the

most common method used for characterizing proteins.
The procedure are relatively rapid and sensitive (require
Figure 2: This cartoon shows a slab of polyacrylamide only μg quantity of protein) and the detection method are
(dark grey) with tunnels (different sized red rings with convenient and sensitive .additionally the use of slab gel
shading to depict depth) exposed on the edge allows the simultaneous analysis of 20 samples in a single
electrophoretic unit. Over the year a number of
Notice that there are many different sizes of tunnels modifications on the basic electrophoretic technique have
scattered randomly throughout the gel. been evolved.Acrylamide gel are formed by polymerizing
acrylamide with a cross linker (bis acrylamide) in the
presence of catalyst (TEMED) and ainitiator (APS) with
the presence of suitable gel buffer(tris). Solutions are
normally degassed prior to polymerization. Oxygen
molecules inhibit polymerization. The rate at which gel
polymerize can be controlled by varying the concentration
of TEMED and APS. The relative proportion of
acrylamide monomer and cross linker (bis acrylamide) can
control the porosity of the gel.Molecules can be separated
by electrophoresis on the basis of charge, size, and shape.
The most widely used system is discontinuous gel
electrophoresis, where the gel is composed 3⁄4separating
gel and 1⁄4 of stacking gel with two buffer system. In
normal gel the sample are loaded directly on the top of the
gel. In this case the sharpness of the protein produced in
Figure 3: This is a top view of two selected tunnels (only the gel will be as broad as possible. This problem can be
two are shown for clarity of the diagram). overcome by polymerizing a short stacking gel on the top
of the separating gel.
These tunnels extend all the way through the gel, but they
meander through the gel and do not go in straight lines.
Notice the difference in diameter of the two tunnels.
4. Principle
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) is
Now we are ready to apply the mixture of denatured
probably the world’s most widely used biochemical
proteins to the gel and apply the current (figure 4).
method. In the early 60's scientists first appreciated the
Because of their small size, they move faster since they
utility of polyacrylamide gels as a convenient and versatile

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Impact Factor (2012): 3.358
alternative to starch gels [3, 4], thus developing mercaptoethanol + 150 mg SDS and a pinch of
polyacrylamide gel electrophoresis or PAGE. The bromophenol blue.
inclusion of ionic detergent Sodium Dodecyl Sulphate 8. Staining solution: -dissolved 200mgcoomassie
(SDS) to the gel and the sample was an important addition brilliant blue R 250 in 50ml methanol/ethanol, 7ml
to this work. Shapiro et al. were one of the first to make acetic acid and 43ml distilled water & filter it.
use of this approach [5]. Laemmli showed that proteins 9. De-staining solution:- add 7ml acetic acid to 30ml
could be reliably fractionated by SDS-PAGE, which he methanol/ethanol and 63ml distilled water
described in a figure legend in a Nature paper [2]. 10. Vertical slab-gel electrophoresis equipment.
11. Acrylamide mixture (10%) for 25 ml of resolving
SDS-polyacrylamide gel electrophoresis involves the gel:-9.9 ml, distilled water + 8.3ml, 0% acrylamide +
separation of protein based on their size. By heating the 6.3ml, 0.5 M tris-HCl + .25ml, 10% SDS + .25ml,
sample under denaturing and reducing condition, protein 10% APS +.01 ml, TEMED.
become unfolded and coated with SDS detergent 12. 5% stacking gel for 5 ml:- 3.04ml, distilled water +
molecules, acquiring a high negative charge that is 0.83ml, 30% acrylamide + .63ml, 0.5 M Tris –
proportional to the length of the polypeptide chain. When HCl(pH-6.8) + 0.05ml, 1% SDS +0.05ml, APS +
loaded onto a gel matrix and placed in an electric field, the 0.005ml, TEMED.
negatively charge protein molecules migrate towards the
positively charge electrode and are separated by a Tip: gel buffer and self-prepared acrylamide/bis-
molecular sieving effect. After visualization by a protein acrylamide stock solution should be filtered, degassed
specific staining technique, the size of protein can b and stored at 4°C.
estimated by comparison of its migration distance, with
that of a standard of known molecular weight. It is Sample Preparation
possible to blot the separated protein onto a positively
charge membrane and to probe with protein specific Samples may be any material containing proteins or
antibodies in a procedure termed western blotting. nucleic acids. These may be biologically derived, for
example from prokaryotic or eukaryotic cells, tissues,
Western blotting is a technique by which protein can be viruses, environmental samples, or purified proteins. In the
transferred from a polyacrylamide gel to a sheet of case of solid tissues or cells, these are often first broken
nitrocellulose in such a way that a faithful replica of the down mechanically using a blender (for larger sample
original gel pattern is obtained. A wide variety of volumes), using a homogenizer (smaller volumes), by
analytical procedure can then be applied to immobilized sonicator or by using cycling of high pressure, and a
protein, which makes western blotting a powerful tool for combination of biochemical and mechanical techniques –
diagnosing various pathological conditions. In this including various types of filtration and centrifugation –
technique a sheet of nitrocellulose is placed against the may be used to separate different cell compartments and
surface of a SDS-PAGE protein fractionation gel and a organelles prior to electrophoresis. Synthetic biomolecules
current applied across the gel and into the nitrocellulose such as oligonucleotides may also be used as analytes.
where they bind finally by non-covalent forces. The
technique involved three step; protein separating by SDS- The sample to analyze is optionally mixed with a chemical
PAGE, blotting and immune assay. denaturant if so desired, usually SDS for proteins or urea
for nucleic acids. SDS is an anionic detergent that
5. Materials Required denatures secondary and non–disulphide linked tertiary
structures, and additionally applies a negative charge to
1. Acrylamide(30% stock):- dissolved 29.2 g each protein in proportion to its mass. Urea breaks the
acrylamide and 0.8 bis-acrylamide in distilled water hydrogen bonds between the base pairs of the nucleic acid,
and make upto 100ml. Store under dark in amber causing the constituent strands to separate. Heating the
colour bottle at 4°c(can use upto 3 month) samples to at least 60 °C further promotes denaturation [6,
2. Resolving gel/ separating gel buffer pH 8.8, 1.5M 7, 8, 9].
tris-HCl: - dissolved 18.17g tris in 75 ml distilled
water. Adjust to pH 8.8 with 6 N HCl. Adjust the total In addition to SDS, proteins may optionally be briefly
volume to 100ml with distilled water and store at 4°c. heated to near boiling in the presence of a reducing agent,
3. Stacking gel buffer, pH-6.8, and 1.0Mtris-HCl:- such as dithiothreitol (DTT) or 2-mercaptoethanol (beta-
dissolved 3g tris in 40 ml distilled water, adjust to pH mercaptoethanol/BME), which further denatures the
6.8 with 6N HCl. Adjust the total volume to 50ml proteins by reducing disulfide linkages, thus overcoming
with distilled water. Store at 4°C. some forms of tertiary protein folding, and breaking up
4. Electrophoresis buffer pH 8.3:- dissolved 3g tris, quaternary protein structure (oligomeric subunits). This is
14.4 g glycine and 1g SDS in 100ml of distilled water. known as reducing SDS-PAGE.A tracking dye may be
Store at 4°C. added to the solution. This typically has a higher
5. Ammonium per sulphate-initiator 10%:- dissolved electrophoretic mobility than the analytes to allow the
0.1 g APS in 1 ml distilled water. experimenter to track the progress of the solution through
6. TEMED(NNN’N’ Tetramethylenediamine):- the gel during the electrophoretic run.
catalyst.
7. Sample buffer:- 7.25 ml distilled water + 1.25 ml 1. Take 1ml of culture solution of E.coli strain DH5α, in
stacking gel buffer + 1ml glycerol + 0.5 ml β- 1.5 ml of eppendorf’s tube.

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2. Then centrifuge at 12000 rpm for 5 min. 3. Remove the comb from the mould; wash the well with
3. Then discard the supernatant. distilled water.
4. Then add 500 μl of TE in the tube and dissolve the
tube by gentle shaking. Tip: with a marker pen mark the number or the
5. Then add same volume of sample buffer. position of the wells before removing the comb. This
6. Finally heat the sample on boiling bath for 5 minutes aids easy loading of sample.
and then immediately keep on ice.
4. Mount the gel on electrophoretic apertures’.
Preparing acrylamide gels 5. Add electrophoresis buffer to the top and bottom
reservoir of the electrophoretic apparatus.
The gels typically consist of acrylamide, bisacrylamide, 6. Load the sample along with marker protein into the
the optional denaturant (SDS or urea), and a buffer with an wells(20 μl)
adjusted pH. The solution may be degassed under a
vacuum to prevent the formation of air bubbles during Electrophoresis
polymerization. Alternatively, butanol may be added to the
resolving gel (for proteins) after it is poured, as butanol Various buffer systems are used in PAGE depending on
removes bubbles and makes the surface smooth [10]. A the nature of the sample and the experimental objective.
source of free radicals and a stabilizer, such as ammonium The buffers used at the anode and cathode may be the
persulfate and TEMED are added to initiate same or different [12, 13, 14]. An electric field is applied
polymerization [11]. The polymerization reaction creates a across the gel, causing the negatively charged proteins or
gel because of the added bisacrylamide, which can form nucleic acids to migrate across the gel from the negative
cross-links between two polyacrylamide molecules. The electrode (the cathode) towards the positive electrode (the
ratio of bisacrylamide to acrylamide can be varied for anode). Depending on their size, each biomolecule moves
special purposes, but is generally about 1 part in 35. The differently through the gel matrix: small molecules more
acrylamide concentration of the gel can also be varied, easily fit through the pores in the gel, while larger ones
generally in the range from 5% to 25%. Lower percentage have more difficulty. The gel is run usually for a few
gels are better for resolving very high molecular weight hours, though this depends on the voltage applied across
molecules, while much higher percentages are needed to the gel; migration occurs more quickly at higher voltages,
resolve smaller proteins. but these results are typically less accurate than at those at
lower voltages. After the set amount of time, the
6. Procedures biomolecules have migrated different distances based on
their size. Smaller biomolecules travel farther down the
Gel Casting Tray gel, while larger ones remain closer to the point of origin.
Biomolecules may therefore be separated roughly
1. Assemble vertical slab gel apparatus using 1mm spacer. according to size, which depends mainly on molecular
weight under denaturing conditions, but also depends on
Tip:-the plate should be thoroughly cleaned and dried higher-order conformation under native conditions.
before use. However, certain glycoproteins behave anomalously on
SDS gels.
2. Seal the glass plates on 3 sides with 1% agarose.
3. Pour the separating gel/ resolving gel mixture to a level 1. Attach the apparatus to the power supply unit and apply
of approximately 2.5 cm below the glass plates, gently 8 V/Cm for stocking gel (70V) and 15 V/Cm for
layer 250μl of TDW over the gel surface. Allow to resolving gel(150-200V)
polymerize. 2. Electrophoresis is continued until bromophenol blue
reaches the bottom of the gel.
Tips: prepare the solution freshly each time it is 3. Dismantles apparatus and remove gel from between the
required. As soon as ammonium persulphate is added, plates and place in a tray containing distilled water cut a
the gel should be poured quickly before the acrylamide small corner of the gel to indicate the direction of
polymerizes. loading.

4. After polymerization remove water from the top. Coomassie Staining

5. Pour the prepared 5% stacking gel over the resolving
gel/ separating gel. 1. Immersethe gel in 5volume of staining solution and stain
6. Immediately insert a comb and allow polymerizing the for 4 hrs.at room temperature with gentle shacking.
gel. Coomassie brilliant blue R reacts non-specifically with
proteins.
Sample Loading 2. Gently agitate the stained gel in destining solution until
the background becomes clear.
1. Prepared the required volume of sample (100mg protein 3. Store in 7 % acetic acid. Visualize the band in an
per lane) + equal volume of sample buffer. illuminator.
2. Heat the sample in boiling water bath for 5 min to
denature the protein. Immediately keep them on ice to
retain the denature stage.
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SDS-PAGE workflow and illustration of an apparatus (Figure 5)

fractions or dried for autoradiography and permanent

recording.

Advantage of SDS PAGE

PAGE has a high loading capacity; up to 10 micrograms of

DNA can be loaded into a single well (1 cm x 1 mm)
without significant loss of resolution. Polyacrylamide
contains few inhibitors of enzymatic reactions. PAGE is an
ideal gel system from which to isolate DNA fragments for
subcloning and other molecular biological techniques.

Disadvantage of SDS PAGE

Figure 5: Illustration of an apparatus used for SDS PAGE The mobility of the fragments can be affected by base
composition making accurate sizing of bands a problem.
7. Result Polyacrylamide quenches fluorescence, making bands
containing less than 25 ng difficult to visualize with
The sample used in experiment are overnight culture of ethidium bromide staining.
E.coli strain DH5α.we successfully separated the protein
on SDS-PAGE and the protein can be separated on basis of 8. Conclusion
its molecular weight separately by the blotting on a
nitrocellulose paper. SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel
electrophoresis, is a technique widely used in
Chemical ingredients and their roles biochemistry, forensics, genetics and molecular biology to
separate proteins according to their electrophoretic
Polyacrylamide gel (PAG) had been known as a potential mobility (a function of length of polypeptide chain or
embedding medium for sectioning tissues as early as 1964, molecular weight as well as higher order protein folding,
and two independent groups employed PAG in posttranslational modifications and other factors).The SDS
electrophoresis in 1959 [15, 16]. It possesses several gel electrophoresis of samples having identical charge to
electrophoretically desirable features that make it a mass ratios results in fractionation by size and is probably
versatile medium. It is a synthetic, thermo-stable, the world's most widely used biochemical method.
transparent, strong, chemically relatively inert gel, and can
be prepared with a wide range of average pore sizes [17]. Proteins are amphoteric compounds; their net charge
The pore size of a gel is determined by two factors, the therefore is determined by the pH of the medium in which
total amount of acrylamide present (%T) (T = Total they are suspended. In a solution with a pH above its
concentration of acrylamide and bisacrylamide monomer) isoelectric point, a protein has a net negative charge and
and the amount of cross-linker (%C) (C = bisacrylamide migrates towards the anode in an electrical field. Below its
concentration). Pore size decreases with increasing %T; isoelectric point, the protein is positively charged and
with cross-linking, 5%C gives the smallest pore size. Any migrates towards the cathode. The net charge carried by a
increase or decrease in %C from 5% increases the pore protein is in addition independent of its size - i.e. the
size, as pore size with respect to %C is a parabolic charge carried per unit mass (or length, given proteins and
function with vertex as 5%C. This appears to be because nucleic acids are linear macromolecules) of molecule
of non-homogeneous bundling of polymer strands within differs from protein to protein. At a given pH therefore,
the gel. This gel material can also withstand high voltage and under non-denaturing conditions, the electrophoretic
gradients, is amenable to various staining and de-staining separation of proteins is determined by both size and
procedures, and can be digested to extract separated charge of the molecules. Nucleic acids however, remain
negative at any pH used for electrophoresis and in addition
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carry a fixed negative charge per unit length of molecule, [16] Raymond S, Weintraub L. (1959). "Acrylamide gel as
provided by the PO4 group of each nucleotide of the a supporting medium for zone electrophoresis.".
nucleic acid. Electrophoretic separation of nucleic acids Science 130 (3377): 711.
therefore is strictly according to size. doi:10.1126/science.130.3377.711. PMID 14436634.
[17] Rüchel R, Steere RL, Erbe EF (1978). "Transmission-
Acknowledgements electron microscopic observations of freeze-etched
polyacrylamide gels". J Chromatogr. 166 (2): 563–
Authors are thankful to all the Central Inland Fisheries 575. doi:10.1016/S0021-9673(00)95641-3.
research Institute (CIFRI) for ample help and support. We
are also thankful to central library of CIFRI for same. Author Profile

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