Gel electrophoresis native, denaturing&reducingLovnish Thakur
Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation.
It is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels used as support media.
Gels are made by free radical-induced polymerization of acrylamide and N,N’-Methylenebisacrylamide.
It is the most widely used technique of electrophoresis.
Gel electrophoresis native, denaturing&reducingLovnish Thakur
Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation.
It is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels used as support media.
Gels are made by free radical-induced polymerization of acrylamide and N,N’-Methylenebisacrylamide.
It is the most widely used technique of electrophoresis.
PAGE is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels use as the support matrix.
widely used and has very much importance.
COMPLETE PROCEDURE & USES are described in the slide.
RAPD markers are decamer DNA fragments.
RAPD is a type of PCR reaction.
as the name suggest it is a fast method when compared to the traditional PCR medthod.
SNP (Single Nucleotide Polymorphic), SNP mapping, SNP profile, SNP types, SNP analysis by gel electropherosis and by mass spectrometry, SNP effects, single strand conformation polymorphism, SNP advantages and disadvantages and application of SNP profile in drug choice
PAGE is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels use as the support matrix.
widely used and has very much importance.
COMPLETE PROCEDURE & USES are described in the slide.
RAPD markers are decamer DNA fragments.
RAPD is a type of PCR reaction.
as the name suggest it is a fast method when compared to the traditional PCR medthod.
SNP (Single Nucleotide Polymorphic), SNP mapping, SNP profile, SNP types, SNP analysis by gel electropherosis and by mass spectrometry, SNP effects, single strand conformation polymorphism, SNP advantages and disadvantages and application of SNP profile in drug choice
Definition, factors affecting electrophoresis, classification of electrophoresis in general, Iso-electric focusing in detail, IEF and its types (based on ampholytes), step wise procedure of IEF process, Problems involved and their remedies, Capillary iso electric focusing and its types, detection of analytes explained in animation (so watch it in slide show mode), advantages and applications of CIEF.
This presentation consist of all information regarding sodium dodecyl sulphate PAGE. The general information regarding electrophoresis and its main types are also included in this presentation.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
2. • 2-D electrophoresis is a powerful and widely used method for the
analysis of complex protein mixtures extracted from cells, tissues, or
other biological samples.
• It is the method available which is capable of simultaneously
separating thousands of proteins.
• This technique separate proteins in two steps, according to two
independent properties:
First-dimension is isoelectric focusing (IEF), which separates
proteins according to their isoelectric points (pI);
Second-dimension is SDS-polyacrylamide gel electrophoresis
(SDS-PAGE), which separates proteins according to their molecular
weights (MW).
• In this way, complex mixtures consisted of thousands of different
proteins can be resolved and the relative amount of each protein can
be determined.
3. • Each spot on the resulting two-dimensional gel potentially
corresponds to a single protein species in the sample.
• Thousands of different proteins can be separated and information
such as the protein pI, the apparent molecular weight, and the
amount of each protein can be obtained.
• At the very beginning of the 70s, two high-performance
electrophoretic separations of proteins were available: i) zone
electrophoresis of proteins in the presence of SDS, as described in its
almost final form by Laemmli, a technique that instantly became very
popular, and still is, and ii) denaturing isoelecric focusing, as
described by Gronow and Griffith.
• As these two techniques used completely independent separation
parameters, it is not surprising that it was soon tried to couple them.
• Two-dimensional electrophoresis was first introduced by O’Farrell in
1975.
4. Principle:
• In 2D GE proteins are separated as per isoelectric point and protein mass.
• Separation of the proteins by isoelectric point is called isoelectric focusing (IEF).
When a gradient of pH is applied to a gel and an electric potential is applied
across the gel, making one end more positive than the other.
• At all pH values other than their isoelectric point, proteins will be charged. If they
are positively charged, they will be pulled towards the negative end of the gel
and if they are negatively charged they will be pulled to the positive end of the
gel. The proteins applied in the first dimension will move along the gel and will
accumulate at their isoelectric point; that is, the point at which the overall
charge on the protein is 0 (a neutral charge).
• In separating the proteins by mass, the gel treated with sodium dodecyl sulfate
(SDS) along with other reagents (SDS-PAGE in 1-D). This denatures the proteins
(that is, it unfolds them into long, straight molecules) and binds a number of SDS
molecules roughly proportional to the protein's length. Because a protein's
length (when unfolded) is roughly proportional to its mass, Since the SDS
molecules are negatively charged, the result of this is that all of the proteins will
have approximately the same mass-to-charge ratio as each other.
5. • In addition, proteins will not migrate when they have no charge (a
result of the isoelectric focusing step) therefore the coating of the
protein in SDS (negatively charged) allows migration of the proteins in
the second dimension.
• In the second dimension, an electric potential is again applied, but at
a 90 degree angle from the first field. The proteins will be attracted to
the more positive side of the gel (because SDS is negatively charged)
proportionally to their mass-to-charge ratio.
• The gel therefore acts like a molecular sieve when the current is
applied, separating the proteins on the basis of their molecular
weight with larger proteins being retained higher in the gel and
smaller proteins being able to pass through the sieve and reach lower
regions of the gel.
7. 1. Sample preparation
2. Isoelectric focusing (first dimension)
3. SDS-PAGE (second dimension)
4. Visualization of proteins spots
5. Identification of protein spots
Steps in 2D-Gel Electrophoresis
8. 1. Sample preparation
• Must break all non-covalent protein-protein, protein-DNA, protein-
lipid interactions, disrupt S-S bonds
• Must prevent proteolysis, accidental phosphorylation, oxidation,
cleavage, deamidation
• Must remove substances that might interfere with separation
process such as salts, polar detergents (SDS), lipids,
polysaccharides, nucleic acids
• Must try to keep proteins soluble during both phases of
electrophoresis process
9. Sample preparation……….
• Protein Solubilization
– 8 M Urea (neutral chaotrope)
– 4% CHAPS (zwitterionic detergent)
– 2-20 mM Tris base (for buffering)
– 5-20 mM DTT (to reduce disulfides)
– Carrier ampholytes or IPG buffer (up to 2% v/v) to enhance
protein solubility and reduce charge-charge interactions
• Protease inhibitors
– PMSF(PhenylmethaneSulfonyl Flouride), Pefabloc, EDTA,,
leupeptin, Aproteinin, Pepstatin
• Contaminant removal
– Filtration, Centrifugation, Chromatography, Solvent Extraction
10. 1st dimension: Isoelectric Focusing
• In a pH gradient and under the influence of an electric field, a protein
will move to the position in the gradient where its net charge is zero.
• A protein with a net positive charge will migrate toward the cathode,
becoming progressively less positively charged as it moves through the
pH gradient until it reaches its pI.
• A protein with a net negative charge will migrate toward the anode,
becoming less negatively charged until it also reaches zero net charge.
• If a protein should diffuse away from its pI, it immediately gains charge
and migrates back. This is the focusing effect which allows proteins to
be separated on the basis of very small charge differences.
• The resolution is determined by the slope of the pH gradient and the
electric field strength so, IEF is therefore performed at high voltages
(typically in excess of 1000 V).
• When the proteins have reached their final positions in the pH
gradient, there is very little ionic movement in the system, resulting in
a very low final current.
11. • The original method for first-dimension IEF depended on ampholyte-
generated pH gradients in cylindrical polyacrylamide gels cast in glass
rods or tubes. Now it is replaced by DryStrip gels.
Advantages of DryStrip gels include:
• The first-dimension separation is more reproducible because the
covalently fixed gradient cannot drift.
• Plastic-backed DryStrip gels are easy to handle. They can be picked up at
either end with forceps or gloved fingers.
• The plastic support film prevents the gels from stretching or breaking.
• More acidic and basic proteins can be separated.
• The sample can be introduced into the DryStrip gel during rehydration.
• DryStrip gels eliminate the need to handle toxic acrylamide monomers.
• Immobilized pH gradients and precise lengths ensure high reproducibility
and reliable gel-to-gel comparisons.
12. 1st dimension: Isoelectric Focusing
• Isoelectric Point (pI):
– pH at which a protein has a neutral charge; loss or gain of protons
H+ in a pH gradient (In a pH below their pI, proteins carry a net
positive charge and in a pH above their pI, they carry a net negative
charge)
• Requires very high voltages (10000V)
• Requires a long period of time (10h)
• Degree of resolution determined by slope of pH gradient and electric
field strength
• Uses ampholytes to establish pH gradient
• IPG strips:
– An immobilized pH gradient (IPG) is made by covalently integrating
acrylamide and variable pH ampholytes at time of gel casting,
Stable gradients
13. Components of rehydration solution
• The choice of the rehydration solution for the sample will depend on its
specific protein solubility requirements.
• A typical solution generally contains urea, nonionic or zwitterionic detergent,
DeStreak Reagent or DTT, the appropriate Pharmalyte or IPG Buffer and a
tracking dye.
• Urea solubilizes and denatures proteins, unfolding them to expose internal
ionizable amino acids. Commonly, 8 M urea is used, but the concentration
can be increased to 9 or 9.8 M
• Thiourea, in addition to urea, can be used to further improve protein
solubilization, particularly for hydrophobic proteins . When using both, the
recommended concentration of urea is 7 M and that of thiourea 2 M.
• Detergent solubilizes hydrophobic proteins and minimizes protein
aggregation. The detergent must have zero net charge—use only nonionic
or zwitterionic detergents. CHAPS, Triton X-100, or NP-40 in the range of 0.5
to 4% are most commonly used.
14. Isoelectric Focusing…………….
Rehydrate IPG
strip & apply
protein sample
Place IPG strip
in IEF apparatus
and apply current
Equilibration, reduction
and alkylation
SDS
Urea
DTT
Iodoacetamide
15. • SDS-PAGE is an electrophoretic method for separating polypeptides
according to their molecular weights. The technique is performed in
polyacrylamide gels containing sodium dodecyl sulfate (SDS).
• SDS is an anionic detergent. SDS masks the charge of the proteins
themselves net negative charge per unit mass.
• Besides SDS, a reducing agent such as DTT is also added to break any
disulfide bonds present in the proteins.
• When proteins are treated with both SDS and a reducing agent, the
degree of electrophoretic separation within a polyacrylamide gel
depends largely on the molecular weight of the protein.
• In fact, there is an approximately linear relationship between the
logarithm of the molecular weight and the relative distance of
migration of the SDS-polypeptide complex.
2nd Dimension (SDS-PAGE)
16. • Separation of proteins on basis of MW, not pI
• Requires modest voltages (200V)
• Requires a shorter period of time (2h)
• Presence of SDS is critical to disrupting structure and
making mobility ~ 1/MW
• Degree of resolution determined by %acrylamide &
electric field strength
17. Steps of SDS PAGE
1) Preparing the system for second-dimension electrophoresis
2) Equilibrating the gel(s) in SDS equilibration buffer
3) Placing the equilibrated gel on the SDS gel
4) Electrophoresis
Equilibrating the gels:
• It is important to proceed immediately to gel equilibration, unless
the IPG strip is being frozen (at -60 °C or below) for future analysis.
• Equilibration is always performed immediately prior to the second-
dimension run, never before storage of the DryStrip gels.
• The second-dimension gel itself should be prepared and ready to
accept the DryStrip gel before beginning the equilibration protocol.
18. Equilibration solution components
• The equilibration step saturates the gel with the SDS buffer system required
for the second dimension separation.
• The equilibration solution contains buffer, urea, glycerol, reductant, SDS, and
dye. An additional equilibration step replaces the reductant with
iodoacetamide.
• Equilibration buffer (75 mM Tris-HCl, pH 8.8) maintains the DryStrip gel in a
pH range appropriate for electrophoresis.
• Urea (6 M) together with glycerol reduces the effects of electroendosmosis
by increasing the viscosity of the buffer.
• Glycerol (30%) together with urea reduces electroendosmosis and improves
transfer of proteins from the first to the second dimension.
• Dithiothreitol (DTT) preserves the fully reduced state of denatured,
unalkylated proteins.
• Sodium dodecyl sulfate (SDS) denatures proteins and forms negatively
charged protein-SDS complexes.
• iodoacetamide alkylates thiol groups on proteins, preventing their
reoxidation during electrophoresis.
19. • iodoacetamide also alkylates residual DTT to prevent point
streaking and other silver-staining artifacts .
• Tracking dye (bromophenol blue) allows monitoring of the progress
of electrophoresis.
• The most commonly used buffer system for second-dimension SDS-
PAGE is the Tris-glycine system described by Laemmli.
• This buffer system separates proteins at high pH, which confers the
advantage of minimal protein aggregation and clean separation
even at relatively heavy protein loads.
20. Procedure:
Equilibration
1. Place 2 mL of Equilibration buffer I on each thawed strip in gel tray.
(This step reduces the proteins.)
2. Place the lid on the gel tray. Place tray on shaker and shake gently for 10
minutes.
3. Remove equilibration buffer I from the strips and drain into non-
chlorinated waste under the hood. Hold the flat side (not slanted side)
down to discard buffer and do not touch the strips.
4. Repeat process with equilibration buffer II. (This step alkylates
proteins.)
SDS Running Buffer
1. Rinse out the graduated cylinder specified for SDS running buffer with
MilliQ H2O.
2. Fill to 100 mL with SDS running buffer (10x) and dilute to 1000 mL with
MilliQ H2O.
3. Remove bubbles from top of beaker.
21. Preparation of Gels
1. While tray is shaking, get out the gels. The liquid within the packaging
has sodium azide, so handle with caution. Remove the white strip
and green comb from the gels.
2. Rinse gels with MilliQ H2O from bottle located in gel room. Be sure to
thoroughly rinse the well and the molecular weight marker well.
3. Blot with filter paper squares to remove excess water (Do not touch
surface of gel inside well). Be careful not to disturb molecular weight
marker well. You do not want water in area where strip will be
placed.
Loading of Gels
1. Loosen the lid and heat the overlay agarose in microwave until
melted. Heat in 10 second (or less) increments. ~30 seconds total.
2. Pick the strip up with tweezers on the “+” side of the strip. Slide the
gel strip onto the edge of the gel tray.
3. Dip the strip in SDS running buffer 5 times and then allow excess
buffer to drain off onto a paper towel.
22. 4. Lay down gel and place strip gel side up onto the top of the gel casette with
the ”+” end toward the molecular weight marker well. Leave the strip at
the top aligned above well.
5. With the gel box upright (lean it against a large tip box), add 1 mL of overlay
agarose to well and MW well. Make sure that no bubbles form. If they do,
remove them with the end of a pipette tip.
6. Push the strip down into the overlay agarose. Push one side down 1st and
slowly push other side down at an angle to prevent formation of bubbles.
Make sure the strip lays flat against the gel.
7. Add 2 μL of appropriate molecular weight marker to the molecular weight
well. Add marker as deep as possible in the well to prevent the molecular
weight marker from spilling over into the other area of the gel.
8. Place gel in gel box. Be careful to hold the gel level while placing it in the gel
box.
9. Once agarose is solidified, fill gel box with the SDS running buffer to the fill
line (don’t pour directly on agarose). You want to fill the cassette chambers
with running buffer as well.
23. Gel Box Run
1. Place the lid on the gel box (red to red; black to black).
2. Plug the gel box into the voltage box (red to red; black to blue).
3. Turn on the voltage to 60 Volts for 15 minutes.
4. Turn the voltage up to 200 Volts for the remainder of the run (45-60
minutes).
After the Gel Run
1. Unplug gel box from voltage box.
2. Remove cassette from gel box, drain SDS running buffer.
3. Crack all 4 joints of the cassette with a green comb.
4. Fill plastic bottom of gel container with MilliQ H2O.
5. Place gel in tray by turning cassette upside down and placing gently
into water.
6. Follow the instructions on the wall of the gel room for the
appropriate stain.
24. IPG strip-
pressed down
into the SDS-
PAGE gel
Positive electrode
Negative electrode
Similar mw but
different pI
Similar pI but
different mw
pH 3 4 5 6 7 8 9 10
4. Detection/Visualization
• Coomassie Stain (100 ng to 10 mg protein)
• Silver Stain (1 ng to 1 mg protein)
• Fluorescent (Sypro Ruby) Stain (1 ng & up)
25. In gel digestion
5. Protein Identification
Phosphoimager for
32P and 35S labelled
1D or 2D gels
Fluoroimager for
SYPRO labelled
1D or 2D gels
Densitometer or
Photo Scanner
Imaging
Melanie (http://ca.expasy.org/melanie
ImageMaster 2D (Amersham)
PDQuest (BioRad )
Analysis
Excision of spots
MSMS/MS
26. Protein detection and image analysis
• This step plays a crucial role, as i) only what is detected can be further
analyzed and ii) quantitative variations observed at this stage are the
basis to select the few spots of interest, in comparative studies, that
will be the only ones processed for further analysis with mass
spectrometry.
• Detection with organic dyes can be summarized in one single process,
colloidal Coomassie Blue staining, which has really become a
reference standard.
• Although the sensitivity is moderate and homogeneity are good and
compatibility with mass spectrometry is excellent.
• Silver staining is much more sensitive but less homogeneous,
because of its delicate mechanism, and its compatibility with mass
spectrometry is problematic.
• The consequence of the presence of formaldehyde at the image
development step, formaldehyde-free silver staining protocols have
been recently proposed.
27. • Protein detection by fluorescence give good sensitivity and also good
compatibility with mass spectrometry .
• Other modes of detection are environment-sensitive probes,
noncovalent binding and covalent binding.
• The use of chemically related, reactive fluorescent probes differing
mainly by their excitation and emission wavelengths allows to perform
multiplexing of samples on 2D gels.
• This multiplexing process solves in turn two difficult problems in the
comparative analysis of gel images, namely the assignment of small
positional differences and taking into account moderate quantitative
changes.
28. • Steps in proteome informatics for 2-DE are:
Image acquisition: This prepares each raw acquisition for subsequent
comparative analysis. After scanning, the images are pre-processed by
cropping (manual delineation), noise suppression, and background
subtraction (e.g., with mathematical morphology or smooth
polynomial surface fitting).
An image capture device is required, for which there are three main
categories:
• Flatbed scanner: This mechanically sweeps a standard charge-coupled
device (CCD) under the gel and can be used to obtain 12–16 bits of
greyscale or colour densitometry from visible light stains. Noise can be
an issue due to size and cooling restrictions. Calibration is often
required to provide linearity. flatbed scanners are typically the least
expensive offerings. Examples: ImageScanner (GE Healthcare),
ProteomeScan (Syngene) and GS-800 (Biorad).
29. • CCD camera: Since the sensor is fixed, its greater size and cooling
provides a dramatic improvement in noise.
Different filters and transillumination options allow a wide range of
stains to be imaged, including visible light, fluorescent, reverse,
chemiluminescent, and radioactive signals.
However, the fixed sensor limits image resolution. Examples: LAS (Fuji
Photo Film), ImageQuant (GE Healthcare), Dyversity (Syngene),
BioSpectrum2D (UVP) and VersaDoc (Biorad).
• Laser scanner: Photomultiplier detectors are combined with laser
light and optical or mechanical scanning to pass an excitation beam
over each target pixel. While slower than CCD cameras, spatial
resolution is excellent. FLA (Fuji Photo Film), Typhoon (GE Healthcare)
and Pharos FX (Biorad).
30. Conventional analysis (Spot Detection ≫ Spot Matching): Each
protein spot is delineated and its volume quantified. The spots are
segmented first by the watershed transform, where spots are
slowly immersed in water. Point pattern matching is then
employed to match the spots between gels, which finds the
closest spot correspondence between a point pattern (source spot
list) and a target point set (reference spot list).
Image-based analysis (Gel alignment ≫ Consensus Spot Modelling):
With current techniques, a “reference” gel is chosen and the other
“source” gels are aligned to it in pair-wise fashion. A similarity
measure which quantifies the quality of alignment between the
warped source gel and the reference gel. The aim is to
automatically find the optimal transformation that maximizes the
similarity measure. Spot detection is then performed on an image,
which is then propagated to each individual gel for spot
quantification.
31. Differential analysis : At this stage, we have a list of spots, and for
each spot, a quantified abundance in each gel. The abundances
are first normalized to remove systemic biases between gels and
between channels in DIGE gels. Variance stabilization can then be
employed to remove the dependence between the mean
abundance of a protein and its variance. Significance tests are
then performed to obtain p-values for rejecting the null
hypothesis that the mean spot abundance between groups is
unregulated.
Advanced techniques : Since multiple hypothesis testing leads to a
large number of false positives, it is essential to control the False
Discovery Rate (FDR). The FDR is the estimated percentage of false
positives within the detected differential expression rather than
within the set of tests as a whole. Power analysis is also estimates
the false negative rate that determines the optimal sample size
needed to detect a specific fold change to a particular confidence
level.
32. Applications
• The proteomics analysis reported here shows that a major cellular response to
oxidative stress is the modification of several peroxiredoxins. An acidic form of
the peroxiredoxins appeared to be systematically increased under oxidative
stress conditions due to post transcriptional modifications.
• Peroxiredoxin 2 and 3 spots in Jurkat cells. cells were separated by two-
dimensional electrophoresis. The peroxiredoxin spots (indicated by arrows)
were identified by mass spectrometry.
• The cells were either control cells (A) or cells treated with 75 M BHP for 1 h
(B). increase in the acidic peroxiredoxin spots under oxidative stress, and the
corresponding decrease in the basic spot under BHP treatment.
1.
33. • Peroxiredoxin spots under various cell
injury conditions.
• The peroxiredoxin spots (indicated by
arrows) were identified by mass
spectrometry. The cells were cultured
under normal conditions (A), submitted
to oxidative stress with 75 M BHP for 30
min (B) or 14 milliunits/ml glucose
oxidase for 18 h (C), or treated with 1
M daunomycin for 18 h (D). The
increase in the acidic spots is correlated
with oxidative stress.
34. Protein Identification by Mass Profile
Fingerprinting
• Due to the high resolution of 2D gels, very simple and cheap MS process can
be used to identify a protein from a 2D gel.
• For example, the old peptide mass fingerprinting method, which is fairly
cheap, fast, and can be carried out on low-price TOF MS, works only with 2D
gel-separated proteins, and will never work with any other technique of less
resolving power.
• We can identify proteins at the sub-microgram level without
sequence determination by chemical degradation.
• The protein, usually isolated by one- or two-dimensional gel
electrophoresis, is digested by enzymatic or chemical means and the
masses of the resulting peptides are determined by mass
spectrometry.
• The resulting mass profile, i.e., the list of the molecular masses of
peptides produced by the digestion, serves as a fingerprint which
uniquely defines a particular protein.
• This fingerprint may be used to search the database of known
sequences to find proteins with a similar profile. This provides a rapid
and sensitive link between genomic sequences and 2D gel
electrophoresis mapping of cellular proteins.
35. 2D gel-based proteomics is widely used in areas where large
series of samples are the norm, for example in toxicology
• 2-DE is used to find an association between decreased calcium-
binding protein (calbindin-D 28 kDa), urinary calcium wasting and
intratubular corticomedullary calcifications in rat kidney .
• They show that in dogs and monkeys, which are generally devoid of
cyclosporine A (CsA)-mediated nephrotoxicity b/c renal calbindin
levels not affected by the CsA treatment
• whereas in CsA-treated human kidney-transplant recipients with renal
vascular or tubular toxicity, a marked decrease in renal calbindin-D 28
kDa protein level was found in most of the kidney biopsy sections.
• It suggest that calbindin is a marker for CsA-nephrotoxicity.
• The discovery of calbindin-D 28 kDa being involved in CsA toxicity has
evolved from the application of 2-DE and has not been reported
previously, proving that proteomics can provide essential information
in mechanistic toxicology.
36. 2D gel-based proteomics in bacterial proteomics
• 2D gel-based proteomics is also widely used in bacterial proteomics, when the
complexity of the sample is low enough.
• (A) Theoretical proteome of B. subtilis showing the distribution of all 4100
predicted proteins according to their isolelectric points and molecular weights.
• (B) B. subtilis master 2-D gel for cytoplasmic proteins which are separated in the
standard pH range 4–7 (right image) and in the alkaline pH range 7–12 (left
image). In the master 2-D gel 519 proteins are labeled that were identified in the
pH range 4–7. In addition, 174 proteins were identified in the narrow range pH
gradients (pH 4–5, 4.5–5.5, 5–6, and 5.5– 6.7) and 52 proteins in the alkaline pH
range 7–12.
• Cytoplasmic proteins were harvested from B. subtilis wild type cells grown in
Belitsky minimal medium at an OD500 of 0.4 and separated by 2-D GE.
37. Use in immunoproteomics
• 2 D GE also used in Immunoproteomics, where it is the immune response of
patients that is probed at a proteomic level.
• 2D GE maps of proteins from Chlamydia trachomatis were probed with sera
from 17 seropositive patients with genital inflammatory disease.
• Immunoblot patterns (comprising 28 to 2 spots, average 14.8) were different
for each patient; however, antibodies against a spot-cluster due to the
chlamydia-specific antigen outer membrane protein-2 (OMP2) were
observed in all sera.
• The next most frequent group of antibodies (15/17; 88%) recognized the
hsp60 like protein, described as immunopathogenic in chlamydial infections.
• A novel outer membrane protein (OmpB) and, interestingly, five conserved
bacterial proteins: RNA polymerase alpha-subunit, ribosomal protein S1,
protein elongation factor EF-Tu, putative stress-induced protease of the HtrA
family, and ribosomal protein L7/L12. These proteins were shown to confer
protective immunity in other bacterial infections.
38. 2D gels in post-translational modifications
• 2D gels are also very appropriate when post-translational
modifications are studied.
• many post translational modifications do alter the pI and/or the
MW of the proteins and thus induce position shifts in 2D gels.
This is true for example for phosphorylation, glycosylation, but
also more delicate modifications such as glutathionylation, or
more forgotten modifications such as protein cleavage.
39. Symptom Possible cause Remedy
No distinct spots are
visible
Sample is insufficient. Increase the amount of sample applied.
Sample contains
impurities that prevent
focusing.
Increase the focusing time or modify
thesample preparation method
The pH gradient is
incorrectly oriented.
The “+” end of the DryStrip is the
acidic end and should point toward the
anode (+).
Detection method was not
sensitive enough.
Use another detection method (e.g.
silver staining instead of Coomassie
blue staining).
Individual proteins
appear as multiple
spots or are missing,
unclear, or in wrong
position
Protein carbamylation. Do not heat any solutions containing
urea above 30 ºC, as cyanate, a urea
degradation the product, will
carbamylate proteins, changing
their pI.
Vertical gap in 2-D
pattern
Impurities in sample. Modify sample preparation
Impurities in rehydration
solution components.
Use only high-quality reagents.
Deionize urea solutions.
Troubleshooting of 2 D Gel Electrophoresis
40. Symptom Possible cause Remedy
Vertical gap in 2-D
pattern
Bubble between DryStrip
gel and top surface of
second-dimension gel.
Ensure that no bubbles are trapped
between the DryStrip gel and the top
surface of the second-dimension gel.
Urea crystals on the
surface of the DryStrip
gel.
Allow residual equilibration solution
to drain from the DryStrip gel before
placing the strip on the second-
dimension gel.
Horizontal stripes
across gel
Impurities in agarose
overlay
or equilibration solution.
Prepare fresh agarose overlay and
equilibration solution
41. Disadvantages of 2D Gel Electrophoresis
• This technique include a large amount of sample handling,
• Limited reproducibility, and a smaller dynamic range than some
other separation methods.
• Difficulty in separation of hydrophobic proteins (Corthals et al,
2000; Wilkins et al, 1998)
• It is also not automated for high throughput analysis.
• Certain proteins are difficult for 2D-PAGE to separate, including
those that are in low abundance, acidic, basic, very large, or
very small.
42. • Separation of proteins by two-dimensional gel electrophoresis (2-DE) coupled with
identification of proteins through peptide mass fingerprinting (PMF) by MALDI-TOF
MS is the widely used technique for proteomic analysis.
• In this work, they investigated the reliability of using raw genome sequences for
identifying proteins. The method is demonstrated for proteomic analysis of
Klebsiella pneumoniae grown anaerobically on glycerol.
• For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis
164 spots were clearly identified as 122 individual proteins.
• 95% of the 164 spots can be successfully identified merely by using peptide mass
fingerprints and a strain-specific protein database (ProtKpn) constructed from the
raw genome sequences of K. pneumoniae. Cross-species protein searching in the
public databases mainly resulted in the identification of 57% of the 66 high
expressed protein spots in comparison to 97% by using the ProtKpn database.
• In conclusion, the use of strain-specific protein database constructed from raw
genome sequences makes it possible to reliably identify most of the proteins from 2-
DE analysis simply through peptide mass fingerprinting.
43. • The serum proteins were separated by two-dimensional electrophoresis (2-
DE); 29 different gene products were identified.
• Proteins represented by 25 spots/spot groups were identified by tandem
nanoelectrospray mass spectrometry (MS), four by matrix-assisted laser
desorption ionization time-of-flight (TOF) MS and one was sequenced by TOF-
TOF technology.
• The identities of four proteins were deduced by similarity to the human
plasma protein database.
• In selected cases, i.e. the immunoglobulins, immunoblotting with specific
antibodies provided additional information about the respective proteins.
• Albumin was detected as the full-length protein and as fragments of various
sizes. Spots representing products of different mass and charge were also
detected for α1-antitrypsin, haptoglobin and transthyretin.
• They are able to identify almost all moderate to high abundance proteins
stained in the serum 2-DE pattern.
44. • Acute phase proteins (APP) have been identified in whey and sera from healthy and mastitis
cows through the proteomic analysis using two-dimensional electrophoresis (2-DE) coupled
with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-
TOF MS).
• Although normal and mastitis serum samples show relatively similar protein composition,
marked differences in expression levels and patterns can be observed.
• Conversely, normal and mastitis whey showed a very different composition, likely due to
extravasation of blood proteins to the mammary gland.
• Different isoforms from the most abundant protein in milk, casein, were detected in both
normal and mastitis whey. Other proteins, such as lactotransferrin, were only detected in
the inflamed animal samples.
• Immunoglobulins showed different patterns but not increased levels in the inflamed whey.
• Also, many cellular proteins in mastitis cow's whey, that were absent from healthy cow's
milk. They are responsible for the great change in composition between normal and mastitis
whey, especially those which exert a biological function related to immune defense.
• Data collected in this work are of interest for gaining information about physiological
changes in protein patterns in different fluids and, the correspondent modifications as result
of an acute phase process in farm.
45. • The serum proteome may be a good tool to identify useful protein biomarkers
for recognising sub-clinical conditions and overt disease in sheep.
• In order to characterize normal protein patterns and improve knowledge of
molecular species-specific characteristics, they generated a two-dimensional
reference map of sheep serum.
• The possible application of this approach was tested by analysing serum
protein patterns in ewes with mild broncho-pulmonary disease, which is very
common in sheep and in the peripartum period which is a stressful time, with
a high incidence of infectious and parasitic diseases. They found overall, 250
protein spots were analyzed, and 138 identified.
• Compared with healthy sheep, serum protein profiles of animals with rhino-
tracheo-bronchitis showed a significant decrease in protein spots identified as
transthyretin, apolipoprotein A1 and a significant increase in spots identified
as haptoglobin, endopin 1b and alpha1B glycoprotein.
• In the peripartum period, haptoglobin, alpha-1-acid glycoprotein,
apolipoprotein A1 levels rose, while transthyretin content dropped.
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Further readings