Protein identification by 2D gels uses a combination of isoelectric focusing (IEF) and SDS-PAGE. In IEF, proteins are separated based on their isoelectric point using an acrylamide gel containing an ampholyte pH gradient established by an electric current. Next, SDS-PAGE further separates the proteins by size. Finally, the proteins are visualized after staining, allowing identification by their position on the 2D gel based on isoelectric point and molecular weight.
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.
Two-dimensional electrophoresis was first introduced by O’Farrell and Klose in 1975
2-DGE is a multi-step separation technique in which proteins are solubilized and separated according to charge (pI) in the first dimension using IEF, followed by size (molecular weight, MW) using SDS-PAGE in the second dimension.
The separated proteins are stained with coomassie or silver stain to produce a two-dimensional protein reference map.
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.
Two-dimensional electrophoresis was first introduced by O’Farrell and Klose in 1975
2-DGE is a multi-step separation technique in which proteins are solubilized and separated according to charge (pI) in the first dimension using IEF, followed by size (molecular weight, MW) using SDS-PAGE in the second dimension.
The separated proteins are stained with coomassie or silver stain to produce a two-dimensional protein reference map.
wo-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first independently introduced by O'Farrell and Klose in 1975.
His Tag Protein Production and PurificationExpedeon
The study of protein regulation, structure, and function relies heavily on the expression and purification of recombinant proteins. Many recombinant proteins are expressed as fusion proteins, meaning that they contain an affinity / epitope tag. A tag is a short sequence of DNA that codes for a specific amino acid, which is frequently inserted into a target gene at the point of coding for expression at either the N or C terminal of the protein required.
gives a very brief info about western blotting procedures, attractive slides, with creative animation effects, i hope this ppt of mine works good for seminar and for educational purposes.
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
Protein microarray Preparation of protein microarray Different methods of arr...naveed ul mushtaq
Protein microarray
Preparation of protein microarray
Different methods of arraying the proteins.FUNCTIONAL PROTEIN MICROARRAYSAnalytical microarrays:-
3.REVERSE PHASE PROTEIN MICROARRAY APPLICATIONS:-
Introduction, Principle, Instrumentation and Applications of SDS-PAGEMohammed Mubeen
The following presentation contains helpful information regarding SDS-PAGE, including the history, introduction, principle, instrumentation, advantages and applications of SDS-PAGE.
wo-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first independently introduced by O'Farrell and Klose in 1975.
His Tag Protein Production and PurificationExpedeon
The study of protein regulation, structure, and function relies heavily on the expression and purification of recombinant proteins. Many recombinant proteins are expressed as fusion proteins, meaning that they contain an affinity / epitope tag. A tag is a short sequence of DNA that codes for a specific amino acid, which is frequently inserted into a target gene at the point of coding for expression at either the N or C terminal of the protein required.
gives a very brief info about western blotting procedures, attractive slides, with creative animation effects, i hope this ppt of mine works good for seminar and for educational purposes.
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
Protein microarray Preparation of protein microarray Different methods of arr...naveed ul mushtaq
Protein microarray
Preparation of protein microarray
Different methods of arraying the proteins.FUNCTIONAL PROTEIN MICROARRAYSAnalytical microarrays:-
3.REVERSE PHASE PROTEIN MICROARRAY APPLICATIONS:-
Introduction, Principle, Instrumentation and Applications of SDS-PAGEMohammed Mubeen
The following presentation contains helpful information regarding SDS-PAGE, including the history, introduction, principle, instrumentation, advantages and applications of SDS-PAGE.
gel electrophoresis # GENETICS AND PTANT BRIDINGsumer06072001
mahabar, barmer, rajasthan
i am plant pathologist
Dr.s.s.rajpurohit
this ppt useful for GPB and biology student . mainly use in assignments in M.Sc. agriculture . this ppt free for student . my YouTube channel: S.S.rajpurohit , comment and contact me for agriculture ppts and other knowledge.
For gel electrophoresis textbook B.D.SINGH
Sodium dodecyl sulphate - Polyacrylamide Gel Electrophoresis (SDS - PAGE) is a technique used for the separation of deoxyribonucleic acid (DNA) ,Ribonucleic acid (RNA) And protein molecules according to their size and electrical charge.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
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.
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.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
Richard's entangled aventures in wonderlandRichard 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.
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.
2. 2D- Gel electrophoresis - combination of two techniques IEF and SDS-PAGE
Proteins are separated based on charge and size.
I dimension – carried out in polyacrylamide gels in narrow tubes in the
presence of urea (8 M urea + anionic detergent)
The denatured proteins are separated according to their isoelectric points.
The gel will be extruded from the tubes by applying slight pressure to one end
then incubated for 15 min in a buffer containing SDS and placed along the
stacking gel of the SDS added gel.
The gel is fixed in place by pouring a molten agarose in electrophoresis buffer
over the gel.
Once the agarose is set,(II dimension) electrophoresis commences and SDS-
bound protein run into the gel, stack and separate according to the size.
3. Isoelectric Focussing
Separation of molecules according to their different isoelectric point.
High resolution – Able to separate proteins that differ in their isoelectric points
by as little as 0.01 of a pH unit.
Horizontal gels on glass plates or plastic sheets.
Separation is achieved by applying potential difference across a gel that contains
a pH gradient.
Gradient in the gel is formed by the introduction of ampholytes - complex
mixture of synthetic polyamino-polycarboxylic acids.
Ampholytes in different pH range
Wide range – 3-10
Narrow range – 7-8
4. pH range is chosen such that the samples being separated will have their pI
values within that range.
Commercially available ampholytes (Bio-lyte and pharmalyte)
Thin layer IEF gels (0.15 mm) thick, prepared using a layer of electrical
insulation tape as the spacer between the gel plates.
IEF is carried out in low % gels to avoid any sieving effect within the gel.
Ampholyte with a suitable pH range and riboflavin will be mixed with the
acrylamide solution and the mixture is poured over the glass plate containing
spacer.
Photopolymerization is performed by placing the gel in front of the bright
light.
5. Photodecomposition of Riboflavin generates a free radical and it initiates the
polymerization. (2-3 h)
Glass plates will be separated so that the gel will be now placed in one of the
glass plate.
pH range is chosen such that the samples being separated will have their pI
values within that range.
Commercially available ampholytes (Bio-lyte and pharmalyte)
Thin layer IEF gels (0.15 mm) thick, prepared using a layer of electrical
insulation tape as the spacer between the gel plates.
IEF is carried out in low % gels to avoid any sieving effect within the gel.
Ampholyte with a suitable pH range and riboflavin will be mixed with the
acrylamide solution and the mixture is poured over the glass plate containing
spacer.
6.
7. Photo polymerization is performed by placing the gel in front of the bright
light.
Photodecomposition of Riboflavin generates a free radical and it initiates the
polymerization. (2-3 h)
Glass plates will be separated so that the gel will be now placed in one of the
glass plate.
Electrode wicks (3 mm thick) strips of wetted filter paper. (Anode –
phosphoric acid & Cathode – Sodium Hydroxide)
This will be laid along the length of each side of the gel and a potential
difference is applied.
Due to this potential difference ampholytes forms the pH gradient between
anode and cathode.
Power is turned-off and samples will be applied by laying on the gel small
squares of filter papers soaked in the sample.
8. A voltage is again applied for about 30 min to allow the sample to
electrophoresis off the paper into the gel.
Later the paper squares will be removed from the gel.
pH region below the pI will be positively charged and will migrate towards
the cathode and vice versa.
As they proceed, the surrounding pH will be steadily increasing and therefore
the positive charge on the protein will decrease correspondingly until
eventually the protein arrives at a point where the pH is equal to its isoelectric
point.
The protein will be in zwitter ion form with no net charge, so further
movement will cease.
Before staining the gel is first placed into10% trichloroacetic acid, which
allows the proteins to get precipitate and the ampholytes will be washed out.
9. The gel is stained with CBB and then destained.
The pI of the particular protein may be determined conveniently by running
a mixture of proteins of known isoelectric point on the same gel.
Used specifically for separating isoenzymes (the different forms of the same
enzyme often differing by one or two amino acid residues)
As the proteins are in native form, the enzymes can be detected in the gel by
washing the unfixed and unstained gel by overlaying with agarose
containing substrate.