SDS Page Gel Electrophoresis

PAGE
Polyacrylamide gel electrophoresis (PAGE) is probably the most common analytical technique used to separate and
characterize proteins. A solution of acrylamide and bisacrylamide is polymerized. Acrylamide alone forms linear
polymers. The bisacrylamide introduces crosslinks between polyacrylamide chains. The 'pore size' is determined by
the ratio of acrylamide to bisacrylamide, and by the concentration of acrylamide. A high ratio of bisacrylamide to
acrylamide and a high acrylamide concentration cause low electrophoretic mobility. Polymerization of acrylamide
and bisacrylamide monomers is induced by ammonium persulfate (APS), which spontaneously decomposes to form
free radicals. TEMED, a free radical stabilizer, is generally included to promote polymerization.

SDS PAGE
Sodium dodecyl sulfate (SDS) is an amphipathic detergent. It has an anionic headgroup and a lipophilic tail. It binds
non-covalently to proteins, with a stoichiometry of around one SDS molecule per two amino acids. SDS causes
proteins to denature and dissassociate from each other (excluding covalent cross-linking). It also confers negative
charge. In the presence of SDS, the intrinsic charge of a protein is masked. During SDS PAGE, all proteins migrate
toward the anode (the positively charged electrode). SDS-treated proteins have very similar charge-to-mass ratios,
and similar shapes. During PAGE, the rate of migration of SDS-treated proteins is effectively determined by
molecular weight.

Below is an example of the procedure for performing discontinuous SDS-PAGE with a 14% separating gel and a 5%
stacking gel.

Materials

PAGE Rigs including glass plates (10 x 20 cm), spacers, comb, and clamps

Power supply

Protein sample

Bio-Rad Laemmli Sample Buffer (contains SDS and either sucrose or glycerol)

2-Mercaptoethanol (reduces disulfide bonds, disrupts protein cross-links)

MW Markers (already prepared in sample buffer)

Gel Cassette Assembly (Bio-Rad Mini Protean 3)

 Clean and completely dry the glass plates, combs, and any other pertinent materials.
 Place a short plate on top of a spacer plate. Insert both plates into the green casting frame on a flat surface.
Be sure that the "legs" of the casting frame are down. Clamp the casting frame and check that the plates are
level on the bottom.
 Put the casting frame into the casting stand.

Preparation of the Gel

 Combine all reagents except the TEMED for the 14% separating gel.

14% Separating Gel Components (4.195 mL)

1.184 milliliters deionized water

1.96 milliliters 30% acrylamide/Bis (Warning: Acrylamide is a neurotoxin. Use gloves, do not

ingest.)

1.0 milliliters 1.5 M Tris, pH 8.8

21 microliters 20% SDS

12 microliters 10% ammonium persulfate

18 microliters TEMED, pH 8.9

 When ready to pour the gel, quickly add the TEMED, mix using a Pasteur pipette, and transfer the
separating gel solution between the glass plates in the casting chamber to about 3/4 inch below the short
plate.

 A small layer of butanol can be added on top of the gel prior to polymerization to straighten the level of the
gel and remove unwanted air bubbles that may be present. Butanol will not mix with the aqueous
separating gel solution. Once the gel has polymerized, the butanol can be removed by absorption with
Kimwipes or filter paper. Rinse the top layer of the gel with dI water prior to pouring the stacking gel.

 Insert the well forming comb into the opening between the glass plates.

 Combine all reagents except the TEMED for the 5% stacking gel.

5.1% Stacking Gel (2.484 mL)

0.4 milliliters deionized water

1.8 milliliters 30% acrylamide/Bis

0.25 milliliters 0.5 M Tris, pH 6.8

12 microliters 20% SDS

17 microliters 10% ammonium persulfate

 5 microliters TEMED

 When ready to pour the gel, quickly add the TEMED, mix using a Pasteur pipette, and transfer the stacking
gel solution between the glass plates in the casting chamber.

 Both the separating and stacking gels should polymerize within six minutes.

 Once the stacking gel has polymerized, the comb can be gently removed. The polymerized gel between the
short plate and spacer plate forms the "gel cassette".

Sample Preparation

 Place some water in a 600 mL beaker and leave on a hot plate to boil. (This can take 15 minutes or more.)

 Meanwhile, add 50 mL of 2-mercaptoethanol to 950 mL of Laemmli sample buffer.

 Combine 10 mL of each protein sample with 20 mL of Laemmli sample buffer plus 2-mercaptoethanol in
microcentrifuge tubes.

 In separate tubes, aliquot 10 mL of MW marker. (MW markers are already prepared in Laemmli sample
buffer.)

 Boil the samples for no more than 5 minutes to fully denature the proteins. Leave the samples at room
temperature until ready to load onto the gel.

Electrophoresis

 Remove the gel cassette from the casting stand and place it in the electrode assembly with the short plate on
the inside.

 Slide the electrode assembly (with the gel cassette) into the clamping frame. Press down on the electrode
assembly while clamping the frame to secure the electrode assembly. This step is important to minimize
potential leakage during the electrophoresis experiment.

 Pour some 1X electrophoresis buffer into the opening of the casting frame between the gel cassettes. Add
enough buffer to fill the wells of the gel. Use a gel loading tip to pipette some buffer into each well to
ensure cleanliness.

 When all wells are sufficiently cleaned, slowly pipette no more than 30 mL of denatured sample or MW
marker into each well. A yellow guide can be placed on top of the electrode assembly to aid in loading the
gel.
 When the gel has been loaded, lower the clamping frame into the electrophoresis tank.

 Fill the region outside of the frame with 1X electrophoresis buffer.

 Cover the tank with the lid aligning the electrodes (black or red) appropriately.

 Connect the electrophoresis tank to the power supply.

 Allow the samples to run at 30 mA until the dye front reaches the bottom of the gel. This can take as long
as 1 hour.
  When electrophoresis is complete, turn off the power supply and disassemble the apparatus.

PAGE (Polyacrylamide Gel Electrophoresis), is an analytical method used to separate components of a

protein mixture based on their size. The technique is based upon the principle that a charged molecule
will migrate in an electric field towards an electrode with opposite sign.The general electrophoresis
techniques cannot be used to determine the molecular weight of biological molecules because the
mobility of a substance in the gel depends on both charge and size. To overcome this, the biological
samples needs to be treated so that they acquire uniform charge, then the electrophoretic mobility
depends primarily on size. For this different protein molecules with different shapes and sizes, needs
to be denatured(done with the aid of SDS) so that the proteins lost their secondary, tertiary or
quaternary structure .The proteins being covered by SDS are negatively charged and when loaded
onto a gel and placed in an electric field, it will migrate towards the anode (positively charged
electrode) are separated by a molecular sieving effect based on size. After the visualization by a
staining (protein-specific) technique, the size of a protein can be calculated by comparing its migration
distance with that of a known molecular weight ladder(marker).

ICP-OES equipment
ICP-OES (Inductively coupled plasma - optical emission spectrometry) is a technique in
which the composition of elements in (mostly water-dissolved) samples can be
determined using plasma and a spectrometer. The technique has been commercially
available since 1974 and thanks to its reliability, multi-element options and high
throughput, it has become a widely applied in both routine research as in more specific
analysis purposes.

The spin-off company B-ware and the GI department use two ICP-OES: primarily the ICP-
OES iCAP 6000 (Thermo Fischer Scientific; only radial measurements possible), and
sometimes the older ICP-OES- Iris Intrepid II (Thermo Fisher Scientific; detection possible
in both radial or axial orientation).

Principle
 The solution to analyze is conducted by a peristaltic pump though a nebulizer into a spray
chamber. the produced aerosol is lead into an argon plasma. Plasma is the forth state of
matter, next to the solid, liquid and gaseous state. In the ICP-OES the plasma is
generated at the end of a quarts torch by a water-cooled induction coil through which a
high frequency alternate current flows. As a consequence an alternate magnetic field is
induced which accelerated electrons into a circular trajectory. Due to collision between
the argon atom and the electrons ionization occurs, giving rise to a stable plasma. the
plasma is extremely hot, 6000-7000 K . In the induction zone it can even reach 10000 K.
In the torch desolvation, atomization and ionizations of the sample takes place. Due to
the thermic energy taken up by the electrons, they reach a higher "excited" state. When
the electrons drop back to ground level energy is liberated as light (photons). Each
element has an own characteristic emission spectrum. By means of an Echelle grating, a
prism, and a focusing mirror these emitted photon in various frequencies are captured
simultaneously on a CCD chip (Charged Coupled Device).

3) Steel Analysis
One major application field of ICP optical emission spectrometry is material analysis. The below
example is an analysis of a steel sample.

3) Hair analysis
Human hair has attracted attention because it is thought to contain a person's health history on
some level and is thought to act as an excretory organ for heavy metal in the body. However,
there are problems because there are few usable samples and knowledge about multiple
elements is required. With simultaneous analysis equipment, we can collect useful information
with a small amount of sample.

Purpose of Making Alloys

Pure metals possess few important physical and metallic properties, such as melting point, boiling point,
density, specific gravity, high malleability, ductility, and heat and electrical conductivity. These properties
can be modified and enhanced by alloying it with some other metal or nonmetal, according to the need.

Alloys are made to:

 Enhance the hardness of a metal: An alloy is harder than its components. Pure metals are generally
soft. The hardness of a metal can be enhanced by alloying it with another metal or nonmetal.
 Lower the melting point: Pure metals have a high melting point. The melting point lowers when pure
metals are alloyed with other metals or nonmetals. This makes the metals easily fusible. This property is utilized
to make useful alloys called solders.
 Enhance tensile strength: Alloy formation increases the tensile strength of the parent metal.
 Enhance corrosion resistance: Alloys are more resistant to corrosion than pure metals. Metals in pure
form are chemically reactive and can be easily corroded by the surrounding atmospheric gases and moisture.
Alloying a metal increases the inertness of the metal, which, in turn, increases corrosion resistance.
 Modify color: The color of pure metal can be modified by alloying it with other metals or nonmetals
containing suitable color pigments.
 Provide better castability: One of the most essential requirements of getting good castings is the
expansion of the metal on solidification. Pure molten metals undergo contraction on solidification. Metals need
to be alloyed to obtain good castings because alloys expand...

Inductively coupled plasma[edit]

Inductively coupled plasma ion source

Inductively coupled plasma (ICP) sources are used primarily for cation analysis of a wide array of
sample types. In this source, a plasma that is electrically neutral overall, but that has had a
substantial fraction of its atoms ionized by high temperature, is used to atomize introduced sample
molecules and to further strip the outer electrons from those atoms. The plasma is usually generated
from argon gas, since the first ionization energy of argon atoms is higher than the first of any other
elements except He, O, F and Ne, but lower than the second ionization energy of all except the most
electropositive metals. The heating is achieved by a radio-frequency current passed through a coil
surrounding the plasma.

Mass Spectrometry
Mass spectrometry is a powerful analytical technique used to quantify known materials, to
identify unknown compounds within a sample, and to elucidate the structure and chemical
properties of different molecules. The complete process involves the conversion of the sample
into gaseous ions, with or without fragmentation, which are then characterized by their mass
to charge ratios (m/z) and relative abundances.

This technique basically studies the effect of ionizing energy on molecules. It depends upon
chemical reactions in the gas phase in which sample molecules are consumed during the
formation of ionic and neutral species.

Basic Principle
A mass spectrometer generates multiple ions from the sample under investigation, it then
separates them according to their specific mass-to-charge ratio (m/z), and then records the
relative abundance of each ion type.

The first step in the mass spectrometric analysis of compounds is the production of gas phase
ions of the compound, basically by electron ionization. This molecular ion undergoes
fragmentation. Each primary product ion derived from the molecular ion, in turn, undergoes
fragmentation, and so on. The ions are separated in the mass spectrometer according to their
mass-to-charge ratio, and are detected in proportion to their abundance. A mass spectrum of
the molecule is thus produced. It displays the result in the form of a plot of ion abundance
versus mass-to-charge ratio. Ions provide information concerning the nature and the structure
of their precursor molecule. In the spectrum of a pure compound, the molecular ion, if
present, appears at the highest value of m/z (followed by ions containing heavier isotopes) and
gives the molecular mass of the compound.

Components
The instrument consists of three major components:

1. Ion Source: For producing gaseous ions from the substance being studied.
2. Analyzer: For resolving the ions into their characteristics mass components according
to their mass-to-charge ratio.
3. Detector System: For detecting the ions and recording the relative abundance of each
 of the resolved ionic species.

In addition, a sample introduction system is necessary to admit the samples to be studied to

the ion source while maintaining the high vacuum requirements (~10-6 to 10-8 mm of
mercury) of the technique; and a computer is required to control the instrument, acquire and
manipulate data, and compare spectra to reference libraries.

Figure: Components of a Mass Spectrometer

With all the above components, a mass spectrometer should always perform the following
processes:

1. Produce ions from the sample in the ionization source.

2. Separate these ions according to their mass-to-charge ratio in the mass analyzer.
3. Eventually, fragment the selected ions and analyze the fragments in a second analyzer.
4. Detect the ions emerging from the last analyzer and measure their abundance with the
detector that converts the ions into electrical signals.
5. Process the signals from the detector that are transmitted to the computer and control
the instrument using feedback.