See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/321727799

SDS-PAGE

Article · January 2017

CITATIONS READS

0 4,488

1 author:

Dyah Wulandari
Suranaree University of Technology
11 PUBLICATIONS   3 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Plant-Microbes Interaction with Mega-plasmid decoration View project

Gelrite Media for Cultivation of Archaea and Thermophilic Acidobacteria View project

All content following this page was uploaded by Dyah Wulandari on 11 December 2017.

The user has requested enhancement of the downloaded file.

 SDS-PAGE

LAB REPORT

By :

1. Sakullapat Homkhao M6030063

2. Pongsatorn Poopisut M5930333
3. Dyah Wulandari D5930166
Results and Discussion:
SDS-PAGE is widely used to analyze the proteins in complex extracts. The system
actually consists of two gels - a resolving gel in which proteins are resolved on the basis of
their molecular weights (MWs) and a stacking gel in which proteins are concentrated prior to
entering the resolving gel. Differences in the compositions of the stacking gel, resolving gel
and electrophoresis buffer produce a system that is capable of finely resolving proteins
according to their molecular weights.

In gel electrophoresis, an electric field is used to move charged molecules through a

matrix of a polymerized substance such as polyacrylamide. The smaller and more highly
charged molecules migrate more rapidly through gels than larger or less charged molecules.
The mobility of a molecule is also affected by the buffer system and the strength of the
electrophoretic field used for the separation. Proteins are much smaller than DNA molecules,
so polyacrylamide gels are used for their separation.

The function of the reagent and buffer:

The polyacrylamide gels used to separate proteins are formed by the chemical polymerization
of acrylamide and a cross-linking reagent, N,N’methylene bisacrylamide. To control the size
of the pores in the gel by adjusting the concentration of acrylamide, as well as the ratio of
acrylamide to bisacrylamide. Raising either the concentration of acrylamide or bisacrylamide,
while holding the other concentration constant, will decrease the pore size of the gel.
Polymerization occurs because of free oxygen radicals that react with the vinyl groups in
acrylamide and bisacrylamide. The oxygen radicals are generated from the catalyst, ammonium
persulfate (APS), when it reacts with a second catalyst, N,N,N’,N’-tetramethylethylenediamine
(TEMED).

Tris HCl 6.8,

Acrylamide gel 5%

Larger pore, lower

ionic strength Tris HCl 8.8,
Acrylamide gel 12%

smaller pore,
higherionic strength
 The solution to denature the proteins with the anionic detergent are sodium dodecyl
sulfate (SDS) and 2-mercaptoethanol. The detergent is sufficient to break the many
noncovalent bonds that stabilize protein folds, and 2-mercaptoethanol breaks any covalent
bonds between cysteine residues. Like other detergents, SDS is an amphipathic molecule,
consisting of a hydrophobic 12-carbon chain and a hydrophilic sulfate group. The SDS
hydrocarbon chain permeates the protein interior and binds to hydrophobic groups, reducing
the protein to a random coil, coated with negatively charged detergent molecules all along its
length.

The stacking and running (resolving) gels have different pore sizes, ionic strengths and pH.
The third component is the electrophoresis buffer (Tris, glycine, SDS), which contains large
amounts of glycine. The ionization state of the glycine is critical to the separation. At neutral
pH, glycine is a zwitterion, with a negatively charged carboxyl group and a positively charged
amino group. Consequently, very little glycine has a negative charge in the chamber buffer or
stacking gel, and significant ionization does not occur until the glycine enters the more alkaline
pH 8.8 environment of the running gel.

The sample buffer used for SDS-PAGE contains a tracking dye, bromophenol blue
(BPB), which will migrate with the leading edge of the proteins being separated on the gel.
The sample buffer also contains glycerol, which allows the protein samples to settle into the
bottom of the gel wells. The gel is vertically positioned in the electrophoresis apparatus and
covered with chamber buffer containing glycine.

Once a voltage is applied, the chloride ions in the sample buffer and stacking gel move
rapidly toward the positive pole, forming the leading edge of a moving ion front. Proteins begin
to migrate at different rates, because of the sieving properties of the gel. Smaller protein-SDS
complexes migrate more quickly than larger proteinSDS complexes (right). Within a certain
range determined by the porosity of the gel, the migration rate of a protein in the running gel
is inversely proportional to the logarithm of its MW.

In our experiments, we will use Simply Blue, a colloidal suspension of Coomassie

Brilliant Blue G-250 to visualize the positions of proteins after electrophoresis is complete.
Brilliant Blue G-250 binds proteins nonspecifically through a large number of ionic and Van
der Waals interactions. In this procedure, gels are rinsed with water to remove the buffer salts
used for electrophoresis and then treated with the colloidal G-250 suspension. Protein bands
appear rapidly, and when necessary, the gels can be destained with deionized water to lower
the gel background. Brilliant Blue staining intensity is considered to be a quantitative
procedure, because with some exceptions, the intensity of a stained band is directly
proportional to the amount of protein in a band.

Results after staining:

- Total cellular protein divide into two fractions, a soluble fraction containing hydrophilic
proteins and an insoluble fraction containing hydrophobic proteins which is based on
differential protein solubility, results in less complex samples and leads to improved
identification of low abundance proteins and a better overall view of the proteome. After
centrifugation, the supernatant containing the soluble hydrophilic proteins is separated from
the pellet containing the hydrophobic/insoluble proteins
- Sample A
Sample A insert with the plasmid pET28- tag with the Green Fluorescence Protein. This
clone will appear green under the UV. To screening the transconjugant use the Kanamycin
antibiotic, because in the plasmid contain Kanamycin antibiotic resistance gene. The plasmid
then transform to the E.coli BL21(DE). This strain of E.coli BL21(DE) specific for protein
expression. From sample A, the overexpress protein appear in 2 bands, which has size around
32.5 & 39.5 kDa. The density of the band mainly thick in the total protein and soluble part,
but insoluble part the band appear thinner.
- Sample B
Sample B insert with the plasmid pET32a. to screening the transconjugant use the
ampicillin antibiotic, because in the plasmid contain ampicillin antibiotic resistance gene. The
plasmid then transform to the E.coli BL21(DE). This strain of E.coli BL21(DE) specific for
protein expression. From sample B, the overexpress protein appear in 1 band, which has size
around 24.5 kDa. The density of the band thick in all total protein, soluble and insoluble part.
- Sample C
Sample C insert with the plasmid pET32- tag with the Red Fluorescence Protein. This
clone will appear red under the UV. To screening the trans conjugant use the ampicillin
antibiotic, because in the plasmid contain ampicillin antibiotic resistance gene. The plasmid
then transform to the E.coli BL21(DE). This strain of E.coli BL21(DE) specific for protein
expression. From sample C, the overexpress protein appear in size around 45 kDa. This size
represent the Ovalbumin protein. The density of the band mainly thick in the total protein and
insoluble part, but soluble part the band appear thinner. This result is correct because for the
total protein consist of mixture the soluble and insoluble part. For the Insoluble part band is
thicker in ovalbumin, because in this experiment we precipitate (denature) the protein by
heating 95C for 5 min. When heated, ovalbumin undergoes a conformational change from its
soluble, serpin structure into an insoluble all-β-sheet structure with
exposed hydrophobic regions. This causes the protein to aggregate and cause the solidification
associated with cooked egg white.
- Sample D
Yeast sample, the band just appear a little bit. Maybe because the protein not induce in
this yeast.

Questions
Cell Culture and Induction

1. What is pET28, pET32 and BL21(DE3)? Why Kanamycin? Why Ampicillin?

Answer

- pET28 is bacterial expression , bacterial expression vector with T7lac promoter,

adds N-terminal His tag, thrombin cleavage site, internal T7 epitope tag, C-terminal His
tag; kanamycin resistance; restriction enzyme cloning.
- pET32 is bacterial expression, production of soluble, active target proteins;
Nterm thrombin cleavage site; Nterm enterokinase cleavage site; a,b,c vary by MCS.
- BL21 is species of bacteria for protein expression.
- Kanamycin is used for bacteria selection that bacteria have pET28.
- Ampicillin is used for bacteria selection that bacteria have pET32.

2. What is IPTG? What is the concentration of the IPTG stock? How much should
we use in step 4?

Answer

- IPTG is a molecular biology reagent. This compound is a molecular mimic of

allolactose, a lactose metabolite that triggers transcription of the lac operon, and it is
therefore used to induce protein expression where the gene is under the control of the
lac operator.
- IPTG 100 mM concentration stock
- We should use 0.1 ml.

3. What is Tris-HCl, Triton X-100, Lysozyme used for?

Answer

- Tris-HCl used for adjust pH stable.

- Triton X-100 use to lyse cells to extract protein.
- Lysozyme is antimicrobial by destroying the cell wall of bacteria.
4. What are the soluble and insoluble fractions?

Answer

- Soluble that fraction of cells, prepared by disruptive biochemical methods, that

is soluble in water.
- Insoluble that fraction of cells, prepared by disruptive biochemical methods,
that is not soluble in water.

SDS-PAGE

5. What is SDS? What is PAGE? Why do we need SDS?

Answer

- SDS (sodium dodecyl sulfate) is a synthetic organic compound, used in cleaning

procedures, and is commonly used as a component for lysing cells during DNA
extraction, and for denaturing proteins in preparation for electrophoresis in the SDS-
PAGE technique.
- PAGE (polyacrylamide gel electrophoresis) 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.
- Because SDS solubilize cell membrane of bacteria and yeast.

Reference
Gelperin DM, White MA, Wilkinson ML et al. (2005) Biochemical and genetic
analysis of the yeast proteome with a movable ORF collection. Genes Develop 19:
2816-2826.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature 227: 680–685.
Shapiro AL, Viñuela E, & Maizel JV, Jr. (1967) Molecular weight estimation of
polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem
Biophys Res Commun 28: 815–820.
Steinberg TH (2009). Protein gel staining methods: An introduction and overview.
Method Enzymol 463: 542-563.
 Appendix

Table 1. log MW and relative migration distance (Rf) of standard curve

MW Rf logMw
116 0.142857 2.064458
66.2 0.242857 1.820858
45 0.371429 1.653213
35 0.5 1.544068
25 0.657143 1.39794
18.4 0.857143 1.264818
14.4 0.942857 1.158362

2.5

y = -1.028x + 2.1032
2 R² = 0.9625

1.5
log MW

0.5

0
0 0.2 0.4 0.6 0.8 1
relative migration distance (Rf)

Figure 1. protein standard curve

View publication stats