Protein processing and production is often hampered by the formation of aggregates that restrict and complicate
the handling of proteins, antibodies and enzymes. NVoy is designed to minimise the sequential losses in consecutive
protein processing steps which would otherwise dramatically reduce the overall protein yield.
NVoy technology is a quantum leap in protein processing, production and analysis. It uses proprietary NV polymers to enhance protein solubility and stability through the formation of multi-point reversible complexes with proteins without altering their structure.
Long acting injectable microparticle formulation - a new dimension for peptid...Merck Life Sciences
Explore the clinical benefits and applications of sustained release drug delivery with this presentation. Access the findings from a technical feasibility study as well as a case study on sustained release microparticle formulation for a sensitive peptide.
High concentration uf formulation challenges & mitigation strategies by s...Merck Life Sciences
Are you working on a high concentration mAb subcutaneous formulation? Join us to find out how to overcome buffer offsets in the formulation UF-DF step.
In this webinar, you will learn:
• High concetnration UF-DF optimization
• Scaling up the UF-DF step
• Models to explain buffer offsets
• Process Development to mitigate buffer offsets
Tangential flow filtration (TFF) using ultrafiltration membranes is commonly used for the final formulation and concentration of monoclonal antibodies. Injection of liquid formulations is often the preferred method of administration for these therapeutics. In cases where high dosages are required, manufacturers frequently target high concentration liquid formulations which can result in high viscosity solutions. Highly concentrated protein solutions have challenges associated with formulation. This talk presents the formulation challenges that are associated with plausible risk mitigation strategies to overcome such formulation challenges.
Tools for improved Protein Mass Spec Sample preparation by PromegaMourad FERHAT, PhD
Proteases, glycosidases, antibody characterization tools, IdeS, IdeZ, mass spec reference protein digests, and new solutions for mass spectrometry analysis. Découvrez nos solutions pour vos analyses en spectrométrie de Masse.
Mourad FERHAT, Ph.D
Chef de Produit Analyse cellulaire
mourad.ferhat@promega.com
Promega France
This presentation reviews the results of a study in which the authors investigated the effects of poly-diallydimethylammonium chloride (pDADMAC) flocculation and clarification on the performance and longevity of protein A resin.
To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: http://www.emdmillipore.com/mlab
The first step in sample preparation is isolating proteins from their source. Usually, proteins are isolated from cells or tissues via lysis. Lysis breaks down the cell membrane to separate proteins from the non-soluble parts of the cell. A number of lysis buffers can be used to prepare samples for western blotting.
NVoy technology is a quantum leap in protein processing, production and analysis. It uses proprietary NV polymers to enhance protein solubility and stability through the formation of multi-point reversible complexes with proteins without altering their structure.
NVoy technology is a quantum leap in protein processing, production and analysis. It uses proprietary NV polymers to enhance protein solubility and stability through the formation of multi-point reversible complexes with proteins without altering their structure.
NVoy technology is a quantum leap in protein processing, production and analysis. It uses proprietary NV polymers to enhance protein solubility and stability through the formation of multi-point reversible complexes with proteins without altering their structure.
Long acting injectable microparticle formulation - a new dimension for peptid...Merck Life Sciences
Explore the clinical benefits and applications of sustained release drug delivery with this presentation. Access the findings from a technical feasibility study as well as a case study on sustained release microparticle formulation for a sensitive peptide.
High concentration uf formulation challenges & mitigation strategies by s...Merck Life Sciences
Are you working on a high concentration mAb subcutaneous formulation? Join us to find out how to overcome buffer offsets in the formulation UF-DF step.
In this webinar, you will learn:
• High concetnration UF-DF optimization
• Scaling up the UF-DF step
• Models to explain buffer offsets
• Process Development to mitigate buffer offsets
Tangential flow filtration (TFF) using ultrafiltration membranes is commonly used for the final formulation and concentration of monoclonal antibodies. Injection of liquid formulations is often the preferred method of administration for these therapeutics. In cases where high dosages are required, manufacturers frequently target high concentration liquid formulations which can result in high viscosity solutions. Highly concentrated protein solutions have challenges associated with formulation. This talk presents the formulation challenges that are associated with plausible risk mitigation strategies to overcome such formulation challenges.
Tools for improved Protein Mass Spec Sample preparation by PromegaMourad FERHAT, PhD
Proteases, glycosidases, antibody characterization tools, IdeS, IdeZ, mass spec reference protein digests, and new solutions for mass spectrometry analysis. Découvrez nos solutions pour vos analyses en spectrométrie de Masse.
Mourad FERHAT, Ph.D
Chef de Produit Analyse cellulaire
mourad.ferhat@promega.com
Promega France
This presentation reviews the results of a study in which the authors investigated the effects of poly-diallydimethylammonium chloride (pDADMAC) flocculation and clarification on the performance and longevity of protein A resin.
To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: http://www.emdmillipore.com/mlab
The first step in sample preparation is isolating proteins from their source. Usually, proteins are isolated from cells or tissues via lysis. Lysis breaks down the cell membrane to separate proteins from the non-soluble parts of the cell. A number of lysis buffers can be used to prepare samples for western blotting.
NVoy technology is a quantum leap in protein processing, production and analysis. It uses proprietary NV polymers to enhance protein solubility and stability through the formation of multi-point reversible complexes with proteins without altering their structure.
NVoy technology is a quantum leap in protein processing, production and analysis. It uses proprietary NV polymers to enhance protein solubility and stability through the formation of multi-point reversible complexes with proteins without altering their structure.
NVoy technology is a quantum leap in protein processing, production and analysis. It uses proprietary NV polymers to enhance protein solubility and stability through the formation of multi-point reversible complexes with proteins without altering their structure.
Proteins and peptides have received increased interest in the current drug therapies
Recently, approved recombinant protein therapeutics have been developed to treat a wide variety of clinical indications, including cancers, exposure to infectious agents, autoimmunity/ inflammation and genetic disorders.
Their high potency and selectivity.
Their low accumulation in tissues.
They have potentially lower toxicity than the small drug molecules.
Provide abroad range of targets, which could represent a basis for personalized medication.
Guide to Molecular Cloning - Download the GuideQIAGEN
Molecular cloning can be sometimes tricky with significant challenges involved. Overcome the challenges with the essential knowledge and tips for successful cloning.
Sr no Contents
1 Introduction
2 Advantages and disadvantages
3 Types of nanoparticle
4 Classification of Nanoparticle
5 Polymers used in nanoparticles
6 Method of preparation
7 Evaluation of nanoparticles
8 Application of nanoparticles
9 References
Nanoparticles is derived from the Greek word Nano means extremely small.
Nanoparticles are sub Nano sized colloidal drug delivery systems .
Particle size ranges from 10-1000 nm in diameter .
They are made up of natural, synthetic or semi synthetic polymers carrying drugs or proteinaceous substances, i.e. antigen(s) .
Drugs are entrapped either in the polymer matrix as a particulates or solid solutions or may be bound to particle surface by physical adsorption or by chemical reaction.
Drug can be added during preparation of nanoparticles or to the previously prepared nanoparticles
Nanoparticles can act as controlled release system depending on their polymeric composition.
As a targeted drug carrier nanoparticles reduce drug toxicity
Less amount of dose required.
They enhance aqueous solubility of poorly soluble drug therefore increase its bioavailability, therapeutic efficacy and Reduces side effects.
Nanoparticles can be administer by various routes including oral, nasal, parenteral, intra-ocular etc.
A) AMPHIPHILIC MACROMOLECULE CROSS-LINKING
B) Polymerization method
C)Polymer precipitation method
Heat cross-linking
Chemical cross-linking
Emulsion chemical dehydration
By Crosslinking in W/O Emulsion
PH-induced aggregation
Counter ion induced aggregation
Emulsion polymerization a)Micellar nucleation and polymerization b)Homogenous nucleation and polymerization)
Dispersion polymerization
Interfacial polymerization
Emulsion solvent evaporation method
Double emulsion and evaporation method
Solvent displacement
Salting out
Nanoprecipitation
ELISA is a well know term that is an abbreviation of Enzyme Linked Immunosorbent Assay. This microplate based technique relies on the use of an antibody that has been linked to an enzyme. In the presence of an appropriate substrate, enzymatic activity produces a color change as the ELISA readout, which can be measured and provides information about the presence and quantity of the target antigen in the sample material.
Electrophoresis is a simple, rapid, and highly sensitive analytical technique to study the properties of proteins and nucleic acids, and has become a principle tool in analytical chemistry, biochemistry, and molecular biology. Polyacrylamide gel electrophoresis (PAGE) can be used to analyze the size, amount, purity, and isoelectric point of polypeptides and proteins. Sodium dodecyl sulfate polyacrylamide discontinuous gel electrophoresis (SDS PAGE) is the most commonly used system whereby proteins become separated strictly by their size, but there are different variations of this technique.
Antibody-oligonucleotide (Ab-Oligo) conjugates have been used in
numerous applications from diagnostics to therapeutics and were
developed through an unmet need for precise and efficient detection of low-abundance proteins. Ab-Oligo conjugates have since played a significant role in enhancing an extensive range of biological techniques that include immunological and proteomic research, biomarker discovery, clinical diagnostics – including point-of-care, as well as other novel techniques. Antibodies can be readily conjugated to oligonucleotides via their amino acid residues, making them suitable for most in vitro applications, as they possess several functional groups.
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.
GELFrEE® 8100 Fractionation System Tech NoteExpedeon
Successful sample preparation is a key step during any analytical
procedure and begins with a defined experimental design. Important steps in sample preparation include proteolytic digestion of proteins into peptide fragments, and peptide fractionation. This is especially important prior to applications such as mass spectrometry (MS).
Antibody-oligonucleotide (Ab-Oligo) conjugates have been used in
numerous applications from diagnostics to therapeutics and were
developed through an unmet need for precise and efficient detection of low-abundance proteins.
Proteomics of small proteins from plant tissuesExpedeon
Small genes and the proteins that they encode can play important biological roles including signaling, development, and mediation of plant-microbe interactions in organisms ranging from bacteria to plants to mammals (Frith et al.; Basrai et al.; Galindo et al.; Hemm et al. 2008, 2010; Kastenmeyer et al.). However, genes that encode proteins containing <100 residues are difficult to identify reliably solely by DNA sequence analysis (Dinger et al.)
Proteomic profiling of fractionated post-myocardial infarctionExpedeon
Acute myocardial infarction remains a leading cause of morbidity and mortality worldwide.Heart failure is the result of adverse remodeling of the collagenous scar that replaces the
damaged myocardium after MI. Markers of LV remodeling can be either identified in the circulation (e.g. serum or plasma) or detected in the heart by imaging technologies or biopsy.
Circular dichroism spectroscopy is an analytical technique used to estimate the secondary and tertiary structure of proteins. This technique can be used to confirm whether structure has been retained during protein processing, but is frequently adversely affected by additives such as solubility enhancers and detergents.
NVoy technology is a quantum leap in protein processing, production and analysis. It uses proprietary NV polymers to enhance protein solubility and stability through the formation of multi-point reversible complexes with proteins without altering their structure.
Proteins and peptides have received increased interest in the current drug therapies
Recently, approved recombinant protein therapeutics have been developed to treat a wide variety of clinical indications, including cancers, exposure to infectious agents, autoimmunity/ inflammation and genetic disorders.
Their high potency and selectivity.
Their low accumulation in tissues.
They have potentially lower toxicity than the small drug molecules.
Provide abroad range of targets, which could represent a basis for personalized medication.
Guide to Molecular Cloning - Download the GuideQIAGEN
Molecular cloning can be sometimes tricky with significant challenges involved. Overcome the challenges with the essential knowledge and tips for successful cloning.
Sr no Contents
1 Introduction
2 Advantages and disadvantages
3 Types of nanoparticle
4 Classification of Nanoparticle
5 Polymers used in nanoparticles
6 Method of preparation
7 Evaluation of nanoparticles
8 Application of nanoparticles
9 References
Nanoparticles is derived from the Greek word Nano means extremely small.
Nanoparticles are sub Nano sized colloidal drug delivery systems .
Particle size ranges from 10-1000 nm in diameter .
They are made up of natural, synthetic or semi synthetic polymers carrying drugs or proteinaceous substances, i.e. antigen(s) .
Drugs are entrapped either in the polymer matrix as a particulates or solid solutions or may be bound to particle surface by physical adsorption or by chemical reaction.
Drug can be added during preparation of nanoparticles or to the previously prepared nanoparticles
Nanoparticles can act as controlled release system depending on their polymeric composition.
As a targeted drug carrier nanoparticles reduce drug toxicity
Less amount of dose required.
They enhance aqueous solubility of poorly soluble drug therefore increase its bioavailability, therapeutic efficacy and Reduces side effects.
Nanoparticles can be administer by various routes including oral, nasal, parenteral, intra-ocular etc.
A) AMPHIPHILIC MACROMOLECULE CROSS-LINKING
B) Polymerization method
C)Polymer precipitation method
Heat cross-linking
Chemical cross-linking
Emulsion chemical dehydration
By Crosslinking in W/O Emulsion
PH-induced aggregation
Counter ion induced aggregation
Emulsion polymerization a)Micellar nucleation and polymerization b)Homogenous nucleation and polymerization)
Dispersion polymerization
Interfacial polymerization
Emulsion solvent evaporation method
Double emulsion and evaporation method
Solvent displacement
Salting out
Nanoprecipitation
ELISA is a well know term that is an abbreviation of Enzyme Linked Immunosorbent Assay. This microplate based technique relies on the use of an antibody that has been linked to an enzyme. In the presence of an appropriate substrate, enzymatic activity produces a color change as the ELISA readout, which can be measured and provides information about the presence and quantity of the target antigen in the sample material.
Electrophoresis is a simple, rapid, and highly sensitive analytical technique to study the properties of proteins and nucleic acids, and has become a principle tool in analytical chemistry, biochemistry, and molecular biology. Polyacrylamide gel electrophoresis (PAGE) can be used to analyze the size, amount, purity, and isoelectric point of polypeptides and proteins. Sodium dodecyl sulfate polyacrylamide discontinuous gel electrophoresis (SDS PAGE) is the most commonly used system whereby proteins become separated strictly by their size, but there are different variations of this technique.
Antibody-oligonucleotide (Ab-Oligo) conjugates have been used in
numerous applications from diagnostics to therapeutics and were
developed through an unmet need for precise and efficient detection of low-abundance proteins. Ab-Oligo conjugates have since played a significant role in enhancing an extensive range of biological techniques that include immunological and proteomic research, biomarker discovery, clinical diagnostics – including point-of-care, as well as other novel techniques. Antibodies can be readily conjugated to oligonucleotides via their amino acid residues, making them suitable for most in vitro applications, as they possess several functional groups.
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.
GELFrEE® 8100 Fractionation System Tech NoteExpedeon
Successful sample preparation is a key step during any analytical
procedure and begins with a defined experimental design. Important steps in sample preparation include proteolytic digestion of proteins into peptide fragments, and peptide fractionation. This is especially important prior to applications such as mass spectrometry (MS).
Antibody-oligonucleotide (Ab-Oligo) conjugates have been used in
numerous applications from diagnostics to therapeutics and were
developed through an unmet need for precise and efficient detection of low-abundance proteins.
Proteomics of small proteins from plant tissuesExpedeon
Small genes and the proteins that they encode can play important biological roles including signaling, development, and mediation of plant-microbe interactions in organisms ranging from bacteria to plants to mammals (Frith et al.; Basrai et al.; Galindo et al.; Hemm et al. 2008, 2010; Kastenmeyer et al.). However, genes that encode proteins containing <100 residues are difficult to identify reliably solely by DNA sequence analysis (Dinger et al.)
Proteomic profiling of fractionated post-myocardial infarctionExpedeon
Acute myocardial infarction remains a leading cause of morbidity and mortality worldwide.Heart failure is the result of adverse remodeling of the collagenous scar that replaces the
damaged myocardium after MI. Markers of LV remodeling can be either identified in the circulation (e.g. serum or plasma) or detected in the heart by imaging technologies or biopsy.
Circular dichroism spectroscopy is an analytical technique used to estimate the secondary and tertiary structure of proteins. This technique can be used to confirm whether structure has been retained during protein processing, but is frequently adversely affected by additives such as solubility enhancers and detergents.
Top down proteomics of soluble and integral membrane proteinsExpedeon
Mitochondria provide important cellular functions including
oxidative phosphorylation, fatty acid biosynthesis, and acting as
gatekeepers to apoptosis.
GELFrEE1 affords rapid mass-based protein separation over a range 10-150 kDa. Here, we demonstrate a multiplexed design enabling increased loading capacity and throughput. We
demonstrate comprehensive analysis of the yeast proteome using GELFrEE coupled to LC-MS/MS analysis.
Identification and characterization of intact proteins in complex mixturesExpedeon
The ability to fully characterize proteins in their intact forms allows thorough biological investigation of the functional importance of changes such as post-translational modifications, protein isoforms/sequence variations, and protease cleavages.
Improved coverage of the proteome using gel eluted liquidExpedeon
It has long been understood that sample fractionation is critically important to generating quality, comprehensive proteomics data. In spite of the continual improvements in speed and sensitivity of mass spectrometers, these instruments are still unable to adequately overcome the enormous challenge
of most biological samples without multiple dimensions of separation prior to mass analysis.
Optimization of experimental protocols for cellular lysisExpedeon
In this project, we have compared existing sample preparation methods for proteomics studies against newly developed FASP method and our in-house developed SDS-TCA protocol. For our
preliminary studies, we have chosen a very well characterized soil microbe Pseudomonas putida.
Characterization of intact antibodies by pre-fractionation using gel electrop...Expedeon
Antibodies represent an important class of proteins due to their central role in the immune response. Moreover, there is an increasing interest in the use of recombinant antibodies as novel drug therapies.
Advances in Capillary Liquid Chromatography for High-Throughput Top Down Prot...Expedeon
Top Down proteomics has benefited greatly from advances in
mass spectrometry instrumentation and database searching,
yet it has been hindered by the lack of robust separation
platforms for intact proteins. Recently, the use of Gel-Eluted
Liquid Fraction Entrapment Electrophoresis (GELFrEE)1,2
followed by capillary liquid chromatography-MS/MS has
enabled hundreds of Top Down identifications from a single
proteome run.
Fast, simple and-cost-effective immunoassay developmet validation and sample ...Expedeon
SPARCL (Spatial Proximity Analyte Reagent Capture Luminescence) is a novel homogeneous proximity assay technology utilizing flash chemiluminescence detection without solid support or wash steps.
Lightning-Link® is an innovative technology that enables direct labeling of antibodies, proteins, peptides or other
biomolecules with only 30 seconds hands-on time.
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.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
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.
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
3. 3
NVoy Polymer Pack
Protection and Release Agent for Protein Processing
KIT CONTENTS
NV10 polymer
STORAGE
Upon receipt store in a cool and dry place. Discard any reagents that show discoloration or evidence of microbial
contamination.
NV10 is stable as supplied. Reconstituted solutions of NV10 can be stored refrigerated for short term (1-7 days) and
for longer term when frozen (6 – 12 months). For further information on storage please refer to question 2 in the FAQ.
HANDLING
This kit is sensitive to small amounts of DNA. Wear gloves at all times and prepare the reaction in a laminar flow hood
or similar device to avoid contamination. Use molecular biology grade clean reagents, sterile reaction tubes and DNA-
free pipette tips. Thaw Polymerase and dNTPs on ice. All other components can be thawed at room temperature.
All chemicals should be considered as potentially hazardous. This material may contain substances or activities
that are harmful to human health. It should not be ingested, inhaled, or brought into contact with skin, and should be
handled with appropriate care in accordance with the principles of good laboratory practice. In case of contact with
skin wash immediately with water. For more specific information, please consult the Material Safety Data Sheets
(MSDS) available on-line at www.expedeon.com.
INTRODUCTION
Protein processing and production is often hampered by the formation of aggregates that restrict and complicate
the handling of proteins, antibodies and enzymes. NVoy is designed to minimise the sequential losses in consecutive
protein processing steps which would otherwise dramatically reduce the overall protein yield.
Utilising NVoy technology is an alternative to the use of detergents, fusion proteins, arginine, chaperones and a range
of other common additives employed to increase protein solubility and enable the handling of proteins in solution.
This NVoy pack contains proprietary carbohydrate polymers, NV polymers, designed to increase the solubility
and stability of proteins whilst preventing aggregation and reducing non-specific binding. NV polymers are linear,
uncharged carbohydrate polymers of around 5kD, derivatised to make them highly amphipathic.. They associate
at multiple points with surface exposed hydrophobic patches of proteins in a dynamic fashion to form multipoint
reversible complexes. Multiple-binding points allow NV polymers to be used at low concentrations relative to
alternative reagents and their size prevents them from entering the protein core and inhibiting normal structural
bonding or blocking catalytic/binding sites. Based on simple carbohydrate polymers, NV polymers are easily
separated from the protein when they are no longer required in solution.
4. 4
NVoy Polymer Pack
Protection and Release Agent for Protein Processing
Impact Areas of NVoy Technology
The impact of NVoy technology can be seen in many areas of protein research including stabilisation, purification,
analysis and crystallisation.
Stabilisation
• Maintain activity over several freeze/thaw cycles
• Prolonged storage at 4ºC for unstable proteins
• Maintain solubility of fusion proteins after “tag” is removed
• Stable formulation of protein / antibody for immobilisation + conjugation
• Maintain soluble proteins that usually require ligand to be present
• Increase concentration of proteins that would otherwise aggregate when concentrated
Purification
• Endotoxin removal
• Minimise protein losses
• Improve purification strategy
• Cleaner protein preparations
Analysis
• Allow full structural characterisation (MS, crystallisation, NMR, CD)
• Use in HTS assays to keep proteins soluble and reduce non-specific binding
• Replacing detergents which are more difficult to remove downstream
Crystallisation
• Controllable crystal growth when rapid formation produces poor crystals
• Concentrate and maintain high protein concentration without the need for any other additives
• Solubilise and stabilise proteins for longer period of time
Refolding
• Simple, effective, generic technique
GENERAL NOTES ON USAGE
1. Generally a 1 mg/ml protein solution can be protected by 1 mg/ml to 5 mg/ml NV10. This corresponds to a 1
to 5 fold excess of NV10 to protein by mass.
2. If protein aggregation occurs, increase the NV10 concentration or decrease the final protein concentration.
If no aggregation is observed it may be possible to decrease the NV10 or increase the protein concentration.
3. The strong release agent will reduce the strength of the interaction between the NV10 and protein molecules.
For a solution containing 1 mg/ml NV10 (0.1% w/w), up to 2.5% (v/v) release agent may be added. This
corresponds to a 25 fold excess of release agent to NV10. Alternatively, organic solvents such as DMSO,
ethanol, or acetone (up to 15% (v/v) can be added), or increased temperature (37 C) can be used to facilitate a
more gentle release. Refrigeration will induce a stronger interaction between protein and NV10.
5. 5
NVoy Polymer Pack
Protection and Release Agent for Protein Processing
PROTOCOLS
A selection of protocols and examples has been included in this manual:
• protein stabilization
• endotoxin removal
• protein concentration
• protein processing & purification
• protein refolding
• NV10 removal
• fusion protein ‘tag-removal’
Recommended Protocol for Protein Stabilisation
Stability is very protein specific, but a general protocol is given below.
• Determine the protein concentration (for example with Expedeon’s BradfordULTRA assay, BCA assay or
absorbance at 280nm).
• Typically a fivefold excess, by mass, of NV10 will protect and stabilise the target protein. For example, use 200
μg/ml NV10 for 40 μg/ml protein.
• The protein solution can be added directly to dry NV10 to get the desired concentration. Alternatively a
concentrated stock solution of up to 50 mg/ml NV10 can be made in an aqueous buffer of choice, which can
then be added to the protein solution.
• Stock solutions containing 2.5 mg/ml NV10 can be stored for 1 week at 4°C or for longer term at -20°C.
Recommended Protocol for Protein Concentration
NVoy technology can be used to minimise protein losses due to aggregation and non-specific binding during
concentration.
• Determine the protein concentration.
• Typically a fivefold excess, by mass, of NV10 will protect and stabilise the target protein e.g. 100 μg/ml NV10
for 20 μg/ml protein.
• Concentrate the protein / NV10 solution to the desired protein concentration.
• NV10 will co-concentrate with the protein in solution to give continuing protection.
EXAMPLE: Concentration of Bovine Serum Albumin
Duplicate samples containing 10 ug/ml BSA in PBS and supplemented with varying concentrations of NV10 were
concentrated tenfold in Vivaspin 2 spin concentrators (5,000 mwco, Hydrosart low protein-binding membrane)
according to the manufacturer’s instructions.
STARTING SOLUTION (1 ML) RECOVERED YIELD (%)
10 μm/ml BSA 46 %
10 µg/ml BSA + 10 µg/ml NV10 60 %
10 µg/ml BSA + 40 µg/ml NV10 85 %
10 µg/ml BSA + 100 µg/ml NV10 90 %
6. 6
NVoy Polymer Pack
Protection and Release Agent for Protein Processing
Recommended Protocol for Protein Refolding
• Solubilise the target protein at 5 mg/ml using chaotropes such as urea and guanidine hydrochloride. Refold the
protein by 20-fold dilution into refolding buffer containing NV10. Incubate overnight at room temperature, add
the protection release solution and perform a further overnight incubation.
• The refolding buffer should contain 0.3 mg/ml – 2 mg/ml concentration of NV10 as a starting point
• If protein aggregation occurs increase the NV10 concentration or decrease the final protein concentration in
the refold.
• The NV10 protection agent will suppress protein aggregation and may allow refolding at final protein
concentrations greater than 0.25 mg/ml. However, the NV10 may also slow down the rate of refolding especially
at 4°C.
• The addition of the protection removal solution is not always required and extended incubation of the refolding
target protein at room temperature may increase yield and allow complete refolding without NV10 release.
• A 5% v/v addition of the protection removal solution will achieve rapid protection release. If the protein is not
completely refolded aggregation may be caused by sudden protein release. If this occurs add the protection
removal solution stepwise with small initial additions or consider performing analysis in the presence of NV10
without release.
• NV10 can be added into the denaturant buffer to assist protein solubilization and then carried over into the
refolding buffer without affecting functionality (e.g. add 20 mg/ml concentration of NV10 to the denaturant
buffer to achieve 1 mg/ml final NV10 concentration in the refolding buffer). Please note that NV10 alone can
not solubilise aggregated protein or inclusion bodies. It is recommended to use chaotropes such as urea or
guanidine hydrochloride in combination with reducing agents.
• Proteins vary widely in properties and requirements. To obtain correct refolding, the inclusion of co-factors,
metal ions and a redox couple in the refolding buffer may be required. When using redox reagents please note
that DTT and other strong reducing agents (potentially carried over from the denaturant solution) will reduce
the oxidized component of the redox couple. We recommend the oxidized/reduced glutathione redox couple at
a final ratio of 2mM reduced to 1 mM oxidized as a good starting point.
Recommended Protocol for Endotoxin Removal
• Bind the target protein to an affinity chromatography or ion exchange media under conditions appropriate to
the target protein.
• Wash the medium with 10 column volumes (CV) of wash buffer containing 2% NV10.
• Wash the medium with 10 CV wash buffer containing 0.2% NV10.
• Wash the medium with 5 CV wash buffer (at 4°C - optional).
• Elute the target protein in elution buffer.
EXAMPLE : Comparison of Endotoxin removal methods.
Target protein was bound to an affinity resin and processed according the NVoy procedure, the Detoxi Gel procedure
(Pierce) and the method by Reichelt et al.1 using Triton X114.
METHOD PROTEIN YIELDS
(% CONTROL)
ENDOTOXIN
EU
ACTIVITY
(% CONTROL)
Control 100 120,000 100
Detoxi Gel 32 3000 55
Triton X114 45 80 32
NVoy 78 None Detected 72
NVoy assisted endotoxin removal was the only method that resulted in a final endotoxin concentration below the
detection limit. Moreover the Novexin method resulted in significantly higher protein recovery and protein activity.
7. 7
NVoy Polymer Pack
Protection and Release Agent for Protein Processing
Protocol for Protein Processing & Purification
• Harvest the Cultured cells and lyse with a buffer containing co-factors and up to 1.0% NV10 (w/v). Do not use
detergents in the lysate buffer.
• If the protein expressed is soluble continue to step 3. If inclusion bodies have formed please refer to the protein
refolding protocol on page 8.
• Clarify the lysate by centrifugation
• Bind the protein on a chromatography support of choice and wash the medium with 10 column volumes of
wash buffer containing up to 1% NV10 (w/v).
• Elute in a buffer of choice which may contain a five fold mass excess NV10 to the expected protein concentration
(e.g. 5mg/ml NV10 for a 1mg/ml protein solution) to enhance protein stability and storage.
By using NV10 over detergents cleaner preparation can be obtained. Moreover, only active protein was obtained
using NV10.
Protocol for Fusion Protein ‘tag-removal’
• Determine the starting protein concentration (for example with Expedeon’s BradfordULTRA assay, BCA assay
or absorbance at 280nm).
• Typically a fivefold excess, by mass, of NV10 will protect the target fusion protein. For example, use 5 mg/ml
NV10 for 1 mg/ml protein.
• Make up the cleavage buffer with the desired NV10 concentration, and use PD10 desalting columns to buffer
exchange the target fusion protein into cleavage buffer containing NV10.
• Continue with tag cleavage according to the standard protocol.
• NV10 associates with the protein in solution and protects the cleaved native protein from aggregation and
instability.
Removal of the maltose binding protein fusion partner (k-MBP) from the kinase, using Factor Xa, results in heavy
aggregation and low yields of the native kinase. By adding NV10 to the cleavage buffer (20 mM Tris.HCl, 75 mM NaCl,
1 mM CaCl2, pH 6.5) aggregation can be significantly reduced, whilst the cleavage reaction remains unaffected.
Recommended Protocol for NV10 Removal
• Bind the target protein to an ion exchange or affinity chromatography media under conditions appropriate to
the target protein.
In most cases the target protein will bind as normal to the resin of choice. If the binding is weaker than expected,
the interaction with the solid phase can be enhanced by using release agents to weaken the interaction between the
protein and NV10 polymer.
• Wash the medium with a minimum of 10 column volumes of binding or wash buffer to remove NV10.
• Elute the target protein using increasing ionic strength, pH gradient or a competing ligand (affinity
chromatography).
8. 8
NVoy Polymer Pack
Protection and Release Agent for Protein Processing
FREQUENTLY ASKED QUESTIONS
Q. Can I make a concentrated solution of NV10?
A. NV10 can be dissolved up to a 50 mg/ml solution in any common aqueous buffer system. However, the NV10
crystallisation will occur faster in more concentrated solution (See Q 3).
Q. Is the NV10 solubility pH dependent?
A. The NV10 solubility is not pH dependent. NV10 can be dissolved in aqueous buffers with pH ranging from 3 to 11.
Q. Can I store a solution of resuspended NV10?
A 1. When NV10 is resuspended in an aqueous buffer the NV10 molecules will slowly crystallise. This will, in time,
lead to a cloudy solution. As a rule of thumb a pure 1 mg/ml NV10 solution is stabile for at least one week at 4 C.
More concentrated solutions will appear cloudy more rapidly. The NV10 crystallisation process only occurs for pure
NV10 solutions. Moreover, despite the undesirable cloudy appearance, a crystallized NV10 solution will retain its
functionality.
A 2. When NV10 is resuspended in an aqueous buffer it is ideally used immediately for protein protection. However,
solutions of resuspended NV10 can also be stored short term (1-7 days) at 4°C and longer term (6–12 months) at
-20°C or -80°C.
Q. Are any purification steps necessary before protein analysis?
A. A 1 mg/ml solution of NV10 is compatible with many analytical techniques.
PROTEIN CONCENTRATION COLUMN CHROMATOGRAPHY
BSA Assay √ √ √ IMAC √ √ √
Bradford Assay* √ √ ION Exhange √ √ √
UV spectroscopy √ √ √ Reversed Phase*** √ √
PROTEIN STRUCTURE PROTEIN ACTIVITY
Biacore √ √ Cell Based Assays √ √ √
Circular Dichroism √ √ √ ELISA Assays √ √ √
Crystallography √ √ FRET assays √ √ √
Electrophoresis √ √ √
Mass spectrometry** √ √ √
NMR √ √
* Use a blank containing NV10. NV10 will give a weak Bradford signal. The assay will show a reduced sensitivity for protein concentrations lower than
0.25 mg/ml. Use Expedeon’s BradfordUltra Assay for higher sensitivity and less background interference.
** Standard C4 zip tip clean up recommended
*** Use of guard column recommended
It may be desirable to carry out protein purification steps before gel-electrophoresis, Bradford analysis and ELISA
since more accurate results may be obtained.
Q. Which is the preferred method to remove NV10 from my protein solution?
A. The recommended purification method is ion-exchange chromatography, or immobilised metal affinity
chromatography (IMAC) if the protein has a histidine tag. Hydrophobic interaction chromatography can be used as a
secondary protein polishing procedure to remove trace levels of NV10.
9. Q. My protein only binds the chromatography resin weakly. Can I improve the binding?
A 1. To enhance the binding to the chromatography resin, release agents may be added to the sample. However,
adding release agents will reduce the protein protection and may lead to higher protein losses on the column.
A 2. The actual protein properties may differ from theoretical protein properties. Try screening different ion exchange
resins and buffers at different pH to find more suitable binding conditions.
Q. Can I use membranes, size exclusion chromatography or desalt resins to separate my protein from NV10
molecules?
A 1. No, these purification strategies are not recommended since NV10 has a large hydrodynamic radius and may
therefore co-elute with your protein in size-based separations.
A 2. To enable size-based purification strategies release agents can be used to weaken the NV10 protein protection.
However, by doing so the protein will no longer be protected, which may result in protein losses.