Environmental stress in Plants

Extreme temperatures, high/low light intensity, draught, flooding, salinity are, to name a few, stressors that plants will be repeatedly subjected to over the course of their life-time. As a result, due to their "sedentary life-style", plants must adapt quickly to their ever-changing environment if they want to survive. Understanding how plants sense, respond and adapt to these stressors is of primordial importance since it has recently been estimated that the temperature increase due to global warming this century will likely exceed previous predictions,[1] hence increasing environmental stress on plants not only by their intensity but also by their frequency. As plant domestication occurred under more favourable conditions than during the early evolution of land plants, crops have been selected on their productivity rather than on their resistance to abiotic stress and with the world population predicted to reach 9.7 billion by 2050, plant scientists face one of the biggest challenge of the future: increasing crop production on less area and with dwindling resources of water.[2]

How do plants respond to draught stress?

It is usually assumed that plant response to stress happens in three different phases.[3]

1. An alarm phase, when plants detect a change in their environment and activate different mechanisms to be able to cope with the change.
2. A resistance phase, where plants adjust their structure and function in order to withstand the stress and repair the damaged caused.
3. If the stressor stops or lessens then the plant may recover and reach a new optimal physiological status. However, if the stress continues or is too intense, then the plant dies.

In the specific case of draught stress, plants are known to reduce photosynthesis by decreasing their leaf area as well as their photosynthesis rate, mainly by inhibiting the CO2 mechanism and by restricting the diffusion of CO2 into the leaf via stomatal closure. As a result of this and because of the low concentration of intracellular CO2, ongoing photosynthetic light reactions may cause the building-up of reduced photosynthetic electron transport components, which can react with molecular oxygen to form highly damaging reactive oxygen species (ROS).[4]

As a result, plants cope with stress by modulating key physiological processes that result in the modification of molecular and cellular processes. This plasticity is mediated by phytohormones such as abscisic acid (ABA), ethylene, cytokinin (CK), gibberellic acid (GA) and auxin as they play important roles in every step of plant development and hence will enable a plant to respond to abiotic stress. For instance, under drought stress, ABA is involved in stomatal closure whereas cytokinins are known to delay leaf senescence and death.[4]

Agrisera antibodies - Working towards a better understanding of environmental stresses in plant research

Development of crops and plants highly resilient to environmental stresses without compromising on yield is one of the many challenges that plant scientists are currently facing. To facilitate research in this area, Agrisera has developed an extensive range of plant antibodies. This includes environmental stress antibodies as well as phytohormone antibodies.

References:

1. Greater future global warming inferred from Earth’s recent energy budget, Brown PT et al, Nature 2017, 552, 45–50

2. Plant abiotic stress challenges from the changing environment; Pereira A, Front Plant Sci., 2016; 7: 1123.

3. Stress Memory and the Inevitable Effects of Drought: a physiological perspective; Fleta-Soriano E et al, Front Plant Sci, 2016,7, 143

4. Plant adaptation to drought stress, Basu S et al, Version 1. F100Res. 2016; 5: 1554.

Written by Magalie Dale

If you like my post why not connect to me on LinkedIn.

Oxidative damage – The damaging effect of Reactive oxygen species ROS

What are ROS?

ROS, Reactive oxygen species, is a generic term to describe a range of oxygen containing radicals such as hydroxyl radical OH., superoxide anion O2-., nitric oxide NO, perhydroxyl radical HO2. and non-radical species such as hydrogen peroxide H2O2 and hypochlorous acid HOCl. They are formed as by-products during normal metabolism processes, primarily in mitochondria, but also as a cellular response to ionising radiations, pollutants, xenobiotics, cytokine and bacterial invasion.1

Oxidative stress - When the concentration of ROS becomes harmful

A certain level of ROS is important for several physiological processes such as wound healing, tissue regeneration and protection from pathogens.2,3 If the concentration of ROS increases, they will be scavenged by enzymatic oxidants (for instance such as SOD, catalase, GPx) or non-enzymatic antioxidants (e.g. vitamin C, vitamin E, transferrin, beta-carotene). However, when ROS are produced too quickly and cells are no longer able to quench them by using suitable antioxidant defences, they become harmful and can cause damage to proteins, lipid molecules of the cell membranes, carbohydrates as well as RNA and DNA. This is referred to as oxidative stress i.e. a state where ROS are overproduced and the rate of clearance via endogenous and exogeneous antioxidants is no longer sufficient to protect cells and tissues from their toxic effects. The biological consequences of oxidative stress include for instance aging, when the levels of ROS remain low but with a gradual increase or cancer when there is a rapid increase in the production of ROS resulting in a high concentration of ROS.

Oxidative stress markers

Unfortunately, ROS radicals are extremely reactive with a short half-life and consequently difficult to use as markers of oxidative stress. Nevertheless, their reactions with lipids, proteins and DNA lead to the formation of stable molecules that can be used as secondary markers of oxidative stress.

1. Protein oxidation: Oxidation of proteins can lead to fragmentation resulting in the loss of their biological activities and the formation of residues such as o-tyrosine, di-tyrosine and dibromo-tyrosine that can be used as markers of oxidative stress.

2. Lipid peroxidation: Polyunsaturated lipid molecules in cell membranes are highly susceptible to reaction with radicals via a chain reaction. This leads to the formation of lipid peroxides that can further decompose into aldehydes such as acrolein, malondialdehyde (MDA), hydroxynonenal (HNE), 4-hydroxy-2-hexenal (HNN), crotonaldehyde (CRA) and adducts such as hexanoyl-lysine (HEL) and 7-ketocholesterol (7KC). Common pathological processes linked to MDA and 4-HNE are Alzheimer’s disease, Parkinson’s disease, cancer, cardiovascular diseases and diabetes.

3. DNA damage: Oxidation of the nucleic acids can lead to the formation of 8-hydroxy 2’-deoxyguanosine, 8-OHdG. Increased levels of 8-OHdG is linked to aging as well as several pathological conditions such as cancer and diabetes and hence represents a useful marker of oxidation stress. 8-OHdG can be easily quantified using ELISA kits from urine or complex samples such as plasma, cell lysates and tissues.

Tools for oxidative stress research

StressMarq has developed an extensive range of products to study oxidative stress. These products are currently available in the UK and Ireland through Newmarket Scientific and include:

• Monoclonal antibodies for lipid peroxidation research.
• DNA damage ELISA kit, quantifying oxidative damage on DNA by measuring 8-OHdG levels
• Dibromo-tyrosine ELISA kit

References:

1. Ray PD et al, Cell Signal. 2012; 24 (5):981-990

2. Onodera Y et al, FEBS Open Bio, 2015; 5: 492–501.

3. Di Meo et al, Oxid Med Cell Longev. 2016; 2016: 7909186.

Written by Magalie Dale

If you like my post why not connect to me on LinkedIn.

Stopping Index Hopping in NGS

Increased mis-assignment of indexes has been shown to occur on Illumina® sequencing instruments that feature a patterned flow cell and exclusion amplification technology. This mis-assignment of indexes, also known as index hopping, index switching or spread of signal, is acknowledged by Illumina on their website.

Illumina® Website (March 2018):

"Index hopping or index switching is a known phenomenon that has impacted NGS technologies from the time sample multiplexing was developed. It causes a specific type of misassignment that results in the incorrect assignment of libraries from the expected index to a different index (in the multiplexed pool).

Index hopping can be seen at slightly elevated levels on instruments using patterned flow cells with exclusion amplification chemistry versus those that do not use patterned flow cells. Libraries with higher levels of free adapters will see higher levels of index hopping."

The phenomenon has been described in a preprint paper (by Rahul Sinha et al.) that suggests the issue might occur during the act of sequencing itself, with free oligos in the pool somehow associating with the fragments being sequenced and causing the fragment to be identified with the index of the free oligo as opposed to it’s true original sequence.

Any single index system may be subject to this phenomenon, but the NEXTFLEX® unique dual index barcodes have been designed to specifically mitigate the index hopping phenomenon. With unique dual index barcodes, if a free oligo associates with a fragment causing a "change" in the index of the read, the other index associated with the fragment will show the read is incorrect and prevent it from being associated with a given sample.

It is important to note however that not all dual index systems would address this problem, as standard dual indexes using a combinatorial approach (using the same first index across sets of a second index) would still lead to the incorrect allocation of some reads as shown below.

In the standard dual indexing example below (combinatorial dual indexing), the fragment was originally indexed with the blue and purple indexes, but it now being identified as indexed with the gold and purple indexes. However because there is also a sample present using the gold and purple index combination, this read is now going to be incorrectly included in that other sample.

However if this same situation occurs with the UNIQUE dual indexes, the resultant contaminant read will not be added to another sample present. The incorrect read will still occur, but this time it isn’t reassigned to another sample present, as no sample contains that combination of indexes. Both indexes are unique so the issue is avoided.

Index mis-assignment can lead to increased false positive rates, which are especially detrimental to sensitive applications.

Multiplexing with NEXTFLEX® unique dual index barcodes significantly increases processing capacity while reducing costs by allowing the user to pool multiple libraries in a single flow cell lane, whilst providing unprecedented data security in sequencing applications.

Written by Rick Bhatt

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NS Reagents Antibodies - Now Available

NS Reagents is the new Newmarket Scientific range of antibodies focussing on Neuroscience and DNA Damage Repair & Methylation, but these are just the areas we are starting with and we have antibodies for other research areas in development, which we will release over the coming months.

Anti-TDP43 (Clone DB9)
Anti-TDP43 (Clone DB9) TAR DNA-binding protein 43 (TDP-43) has been shown to bind both DNA and RNA and has multiple functions in transcriptional repression, pre-mRNA splicing and translational regulation. It belongs to the hnRNP protein family and is highly expressed in the pancreas, placenta, lung, genital tract and spleen. Characterisation of transcriptome-wide binding sites revealed that thousands of RNAs are bound by TDP-43 in neurons.

TDP-43 regulates alternate splicing of the CFTR gene. The resulting aberrant splicing is associated with pathological features typical of cystic fibrosis. Mutations in TDP-43 have been associated with amyotrophic lateral sclerosis, frontotemporal dementia, Parkinson's disease and Alzheimer's disease.

Applications: ELISA ¦ IHC ¦ WB   Host: Mouse   Reactivity: Human


Anti-DNA damage-binding protein 1
DNA damage-binding protein 1 (DDB1) is the large subunit (p127) of the heterodimeric DNA damage-binding (DDB) complex. DDB1 also functions as a core component of the cullin 4 (CUL4) ubiquitin E3 ligase complex, facilitating the binding of substrates to this complex and the ubiquitination of proteins. These factors (ubiquitin ligase substrates) regulate numerous essential processes in the cell including DNA repair (DDB2), DNA replication, chromatin remodelling (Cdt2) and more.

Applications: WB      Host: Rabbit      Reactivity: Human


Details of the full antibody range is available on our website at https://www.newmarketscientific.com/nsreagents

True optimisation of antibodies for immunohistochemistry

Immunohistochemistry (IHC) is often considered something of a "black art". As well as requiring a high degree of knowledge of the techniques involved, it generally requires an element of trial and error to have a given antibody work well with a given tissue and frequently necessitates the use of antibodies and techniques tailored to each individual project or diagnostic application.

This means, there is no one specific protocol for IHC that can be used regularly.

Antibody optimisation for IHC involves a range of tests in order to find an antibodies optimal staining conditions. Each antigen has a preferred method of antigen retrieval such as Heat Induced Epitope Retrieval (HIER) using acidic Citrate or TRIS-EDTA base buffers, as well as an enzymatic retrieval process. However the majority of antigens need an alkaline pre-treatment method for optimal staining. Additionally each antibody has an optimal concentration when used, depending on the affinity of paratope and epitope, as well as the expression level of the antigen.

Antibodies optimised with tissues that express high levels of antigen may prove inadequate when staining tissues with low antigen expression. For this reason it is necessary to optimise each antibody for a variety of tissue types.

Additionally from a clinical perspective, antibodies must be specific with high affinity towards their epitopes, whilst remaining flexible to use and offering good LOT consistency.

This is the basis of our Optibodies range with each antibody optimised for each use.

Optibodies are a range of carefully tested, top quality antibodies for Immunohistochemistry that has been carefully optimised and fine-tuned for both research and clinical IHC using NordiQC recommended control tissues and criteria.

Optibodies ^(TM) from Nordic Biosite - Optimal Antibodies for Optimal Immunohistochemistry

Details of the full Optibodies antibody range are available here.

EpCAM

Her2

PDL1

SYP

HSP70

HSP70s are monomeric proteins that reside in the cytosol of prokaryotes and the cytosol, nuclei, ER, mitochondria and chloroplasts of eukaryotes ⁸. In addition to their intracellular location, HSP70s have been found in the plasma membrane of malignantly transformed cells, on virally / bacterially infected cells and in the extracellular space. Extracellular Hsp70 exists in a free soluble form, complexed to antigenic peptides, or in exosomes ^12,13,14,15. Noteworthy, Hsp70-1 as the most prominent member of the HSP70 family can be detected in the plasma membrane of a large proportion of different tumor entities, but not in the plasma membrane of normal cells/tissues ^16,17,18. Several observations have led to the hypothesis that in tumor cells Hsp70-1 is an integral membrane protein associated with certain membrane lipid components ^{15, 16}.

Fig 1: Ribbon and tube representation of the tertiary Hsp70-1 structure in the presence of ADP.

Heat shock proteins (HSPs) were originally described in the early 1960s by the pioneering work of Ferruccio Ritossa on the fruit fly Drosophila melanogaster ^5,6,7. Expression of HSPs was found as being induced after exposure to different kinds of stress such as heat shock and could be demonstrated subsequently in any cellular organism ⁸. Nevertheless, other stress conditions, including heavy metals, hypoxia, nutrient deprivation and irradiation as well as oxidative and toxic stress, infections and exposure to inflammatory cytokines are also able to induce HSP expression ^{8, 9}. Members of the HSP70 family were identified for the first time as being upregulated in bacteria in response to cellular stress ¹⁰. The painstaking analysis of the limited number of proteins firstly identified by heat shock induction in D. melanogaster and in E. coli led to the finding that DnaK, DnaJ and GrpE were also members of the heat shock class of proteins. In 1984, Bardwell and Craig demonstrated that the E. coli DnaK and the Drosophila 70 kDa heat shock proteins were highly conserved at the sequence level ¹¹. Moreover, hybridization between the DNA of the archaebacterium Methanosarcina barkeri and the HSP70 genes of D. melanogaster, Saccharomyces cerevisiae, and E. coli has been detected, suggesting the existence of Hsp70-related genes in the three “primary kingdoms”: eukaryotes, eubacteria, and archaebacteria¹¹.

The HSP70 Family

The HSP70 family represents the most conserved and best characterized group of HSPs comprising polypeptides whose molecular weights range from 66 – 78 kDa and that are encoded by a multigene family encompassing up to 17 genes and 30 pseudogenes in humans ¹⁹. Functional genes encoding HSP70 proteins map to human chromosomes 6, 14, 21, and at least one other chromosome ²⁰. The most studied genes are HSPA1A and HSPA1B encoding proteins that only differ by two amino acids and are believed as being completely interchangeable proteins. The genes are clustered in the major histocompatibility complex class III region on chromosome 6p21.3.

The HSP70 family constitutes one of the most conserved protein families in evolution. Their members are present in all organisms and subcellular compartments and can be found from archaebacteria and plants to humans. In archaea and eubacteria Hsp70 is referred to as DnaK. In yeasts they are called Ssa, in mammals including humans they are referred to as HspA.

More information is available on the HSP70 website
and antibodies and related products are available by searching on the Newmarket Scientific website.

References:
1. Gribaldo,S. et al. Discontinuous occurrence of the hsp70 (dnaK) gene among Archaea and sequence features of HSP70 suggest a novel outlook on phylogenies inferred from this protein. J. Bacteriol.181, 434-443 (1999). [PubMed]

2. Sharma,D. & Masison,D.C. Hsp70 structure, function, regulation and influence on yeast prions. Protein Pept. Lett.16, 571-581 (2009). [PubMed]

3. Sharma,D. et al. Function of SSA subfamily of Hsp70 within and across species varies widely in complementing Saccharomyces cerevisiae cell growth and prion propagation. PLoS. ONE.4, e6644 (2009). [PubMed]

4. Kampinga,H.H. & Craig,E.A. The HSP70 chaperone machinery: J proteins as drivers of functional specificity. Nat. Rev. Mol. Cell Biol.11, 579-592 (2010). [PubMed]

5. Ritossa,F. Experimental activation of specific loci in polytene chromosomes of Drosophila. Exp. Cell Res.35, 601-607 (1963). DOI: 10.1016/0014-4827(64)90147-8

6. Ritossa,F. New puffs induced by temperature shock, DNP and salicilate in salivary chromosomes of D. melanogaster. Drosophila Information Service37, 122-123 (1963). [Drosophila Information Service]

7. Ritossa,F. A new puffing pattern induced by temperature shock and DNP in Drosophila. Experientia18, 571-573 (1962). DOI: 10.1007/BF02172188

8. Lindquist,S. & Craig,E.A. The heat-shock proteins. Annu. Rev. Genet.22, 631-677 (1988). [PubMed]

9. Jäättelä,M. Heat shock proteins as cellular lifeguards. Ann. Med.31, 261-271 (1999). [PubMed]

10. Craig,E.A. & Gross,C.A. Is hsp70 the cellular thermometer? Trends Biochem. Sci.16, 135-140 (1991). [PubMed]

11. Bardwell,J.C. & Craig,E.A. Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous. Proc. Natl. Acad. Sci. U. S. A81, 848-852 (1984). [PubMed]

12. Bausero,M.A., Gastpar,R., Multhoff,G., & Asea,A. Alternative mechanism by which IFN-gamma enhances tumor recognition: active release of heat shock protein 72. J. Immunol.175, 2900-2912 (2005). [PubMed]

13. Gastpar,R. et al. Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells. Cancer Res.65, 5238-5247 (2005). [PubMed]

14. Lancaster,G.I. & Febbraio,M.A. Exosome-dependent trafficking of HSP70: a novel secretory pathway for cellular stress proteins. J. Biol. Chem.280, 23349-23355 (2005). [PubMed]

15. Vega,V.L. et al. Hsp70 translocates into the plasma membrane after stress and is released into the extracellular environment in a membrane-associated form that activates macrophages. J. Immunol.180, 4299-4307 (2008). [PubMed]

16. Gehrmann,M. et al. Tumor-specific Hsp70 plasma membrane localization is enabled by the glycosphingolipid Gb3. PLoS. ONE.3, e1925 (2008). [PubMed]

17. Schilling,D. et al. Binding of heat shock protein 70 to extracellular phosphatidylserine promotes killing of normoxic and hypoxic tumor cells. FASEB J.23, 2467-2477 (2009). [PubMed]

18. Stangl,S. et al. Targeting membrane heat-shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody. Proc. Natl. Acad. Sci. U. S. A108, 733-738 (2011). [PubMed]

19. Brocchieri,L., Conway de,M.E., & Macario,A.J. hsp70 genes in the human genome: Conservation and differentiation patterns predict a wide array of overlapping and specialized functions. BMC. Evol. Biol.8, 19 (2008). [PubMed]

20. Harrison,G.S. et al. Chromosomal location of human genes encoding major heat-shock protein HSP70. Somat. Cell Mol. Genet.13, 119-130 (1987). [PubMed]

A unique kit for the detection of Dibromo-tyrosine

Dibromo-tyrosine is produced by the oxidative bromination of tyrosine residues. This reaction occurs via eosinophil peroxidase (EPO), an enzyme released by activated eosinophils. Upon activation of eosinophils, a respiratory burst occurs releasing elevated levels of O2 and H202. In the oxidation of tyrosine, EPO utilises H202 to catalyse the peroxidation of physiological levels of bromine found within plasma to generate the brominating reagent hypobromous acid (HOBr) (Ref: 1-5)

Figure 1. Bromination of tyrosine (Ref: 8)

Eosinophils play an immunomodulatory role through their recruitment to host sites of parasitic invasion. EPO levels also contribute to diseases such as asthma, cancers and allergic disorders where cellular activation is found to occur at pathological sites (Ref: 6-10)

Brominated products such as 3,5-dibromo-tyrosine serve as biological markers for in vivo eosinophil-mediated tissue damage which allows for understanding the overall roll oxidative stress has on pathways implicated in diseased states within organisms (Ref: 4) .


About This Assay

This unique kit is part of the StressMarq range. It is a is a competitive ELISA assay that can be used for the quantification of 3,5-dibromo-tyrosine in urine, plasma, and other sample matrices. The assay utilises a dibromo-tyrosine-coated plate and an biotin-conjugated antibody for detection which provides an assay range of 0.078 - 5 μg/mL, with a sensitivity of 0.04 μg/mL. Additional kit highlights are quick incubation times, stable reagents, and an easy to use protocol.

It is important to note that the dibromo-tyrosine antibody used in this assay recognises both free dibromo-tyrosine and brominated residues within a protein. Since complex samples such as plasma, are comprised of mixtures of protein fragments and free 3,5-dibromo-tyrosine, concentrations of 3,5-dibromo-tyrosine reported by ELISA methodology may not coincide with literature values where the free residue is typically measured. This should be kept in mind when analysing and interpreting experimental results.



Assay Overview

Figure 2. Schematic of the dibromo-tyrosine competitive ELISA

Further details for this kit are available here:
Dibromo-tyrosine ELISA Kit Details and Pricing

References:
1. MacPherson, J.C., Comhair, S. A. A., Erzurum, S.C., et al. Eosinophils are a major source of nitric oxide-derived oxidants in severe asthma: characterization of pathways available to eosinophils for generating reactive nitrogen species. J. Immun. 166, 5763-577 (2001).
2. Mayeno, A. N., Curran, A. J., Roberts, R. L., et al. Eosinophils Preferentially Use Bromide to Generate Halogenating Agents. J. Biol. Chem. 264, 5660-5668 (1989).
3. Babior, B. M. Oxygen-dependent microbial killing by phagocytes. N. Engl. J. Med. 298, 659-668 (1978).
4. Wu W., Chen, Y., d’Avignon, A. et al. 3-Bromotyrosine and 3,5-dibromotyrosine are major products of protein oxidation by eosinophil peroxidase: potential markers for eosinophil-dependent tissue injury in vivo. Biochem. 38, 3538-3548 (1999)
5. Kambayashi, Y., Ogino, K., Takemoto, K. et al. Preparation and characterization of a polyclonal antibody against brominated protein. J. Clin. Biochem. Nutr. 44, 95-103 2009
6. Wang J., Slungaard A. Role of eosinophil peroxidase in host defense and disease pathology. Arch. Biochem. Biophys. 445, 256–260 (2006).
7. Kazura, J. W., Fanning, M. M., Blumer, J. L. Mahmoud, A. A. Role of cellgenerated hydrogen peroxide in granulocyte-mediated killing of schistosomula of Schistosoma mansoni in vitro. J. Clin. Invest. 67, 93 (1981).
8. Klebanoff, S. J., Locksley, R. M., Jong, E. C., Rosen, H. Oxidative response of phagocytes to parasite invasion. CIBA Found. Symp. 99: 92 (1983)
9. Gleich, G. J., Ottesen, E. A., Leiferman, K. M., Ackerman, S. J. Eosinophils and human disease. Int. Arc. Allergy Appl. Immunol. 88: 59 (1989).
10. Wardlaw, A. J., Eosinophils in the 1990s: new perspectives on their role in health and disease. Postgrad. Med. J. 70: 536 (1994).

EXO-DNAc-PS: Isolation of circulating and EV-associated DNA

Circulating DNA is emerging as a novel non-invasive tool for patient’s stratification and disease monitoring.

While most of the research has focused on circulating cell-free (cfDNA) or circulating-tumorcell-(CTC)-derived DNA, extracellular vesicle-(EVs)-associated DNA (EV-DNA) is emerging as a third valuable “liquid biopsy” platform.

Genomic single or double-stranded DNA and mitocondrial DNA have been detected in extracellular vesicles. In particular the majority of double-stranded DNA in extracellular vesicles seems to be associated with tumor derived exosomes (Thakur BK et al. 2014; Kalhert et al. 2014 ) where it represents the entire genome of the cancerous tumor from which exosomes were derived.

This discovery indicates the potential of exosomes as a possible diagnostic tool. Exosomes can easily be obtained from a simple blood sample, so combined with an equally simple method of isolation, they may provide an important diagnostic method in the future.


Get genomic DNA from exosomes with the EXO-DNAc Kit.

This kit combines the ability of our DNA-Prep reagent to co-isolate EVs and circulating DNA from biofluids or culture supernatants with a user friendly system for DNA purification. Isolated vesicles are simply lysed with the appropriate lysis buffer and DNA is purified by spin columns and optimised buffers with a fast turnaround time (approximatively 30 minutes).

Then finally, EXO-DNAc provides a DNA concentrator for concentrating the yield (4 fold concentration) and increasing the purity of the DNA to the levels required for digital PCR analysis.

Click here for further information on the Exo-DNAc Kits 

WHITE PAPER: Increasing Ligation Efficiency and Discovery of miRNAs for Small RNA NGS Sequencing Library Prep with Plant Samples.

MicroRNAs (miRNAs) are 18-22 nucleotide long non-coding small RNAs that regulate protein expression and are involved in various cellular process such as development, growth, and physiology. Many studies have shown that miRNA expression in plants is altered by stress-response and environmental changes. Thus, the analysis of miRNA profiles using next generation sequencing can be beneficial to our understanding of stress tolerance of crops and other plants.

Although miRNAs in plants and animals share many similarities, most mature miRNAs in plants contain 2’-O-methylation at the 3’ end. Typically, ligation of a 3’ adapter via the 3’ OH of the miRNA molecule is the first step in small RNA next-generation sequencing (NGS) sequencing library construction. 2’-O-methylation of plant miRNAs reduces ligation efficiency of the 3’ OH, making plant miRNA libraries difficult to generate.

Here, we show that miRNA profiling from bean, wheat, corn, and rice using total RNA inputs can be successfully achieved using the NEXTflex® Small RNA Sequencing Kit v3, without library purification from PAGE gels, and that extending the ligation incubation step increases library yield. Importantly, many putative novel miRNAs were discovered with the NEXTflex® Small RNA Sequencing Kit v3 that were not discovered using other methods. Thus, this protocol allows generation of reduced-bias small RNA libraries from plant total RNA without the tedious step of gel-based size selection, enabling researchers to discover and profile more small RNAs from more samples than with traditional methods.

 

The full white paper is available here:
https://www.newmarketscientific.com/datasheets/Increasing-Ligation-Efficiency-and-Discovery-of-miRNAs-for-Small-RNA-NGS-Sequencing-Library-Prep-with-Plant-Samples.pdf

 

References:
1. Trends in Plant Science, Vol. 17, Issue 4, p196–203, J Cell Physiol. 2016 Feb;231(2):303-13, Curr Opin Plant Biol. 2016 Dec;34:68-76
2. RNA Biology 9:10, 1218–1223; October 2012, The Plant Cell, Vol. 25: 2383–2399, July 2013 3. Nucleic Acids Res, 2011. 39(21): p. e141., RNA, 2011. 17(12): p. 225662., Silence, 2012.
3(1): p. 4., Genome Biol, 2013. 14(10): p. R109.
4. BMC Bioinformatics. 2014; 15(1): 275.

Anti-Human (Cell Membrane Bound) HSP70 Monoclonal

HSP70 is a highly conserved protein that is ubiquitously expressed. It can be found in chloroplasts, eukaryotic cytosol, endoplasmic reticulum, and mitochondria, but also embedded in the cell membrane and in the extracellular space (1,2,3,4). Even though HSP70 is one of the most studied heat shock proteins, the export mechanism and method of membrane insertion are not fully understood. Most proteins in the heat shock family lack a consensus signal for secretion via the ER-Golgi pathway (5).

Researchers have found that HSP70 may be released from cells via exosomes or secretory vesicles (6). Although HSP70 is ubiquitously expressed, there is not much information about its presence on cell surface.

The finding that HSP70 is localized on the cell surface of cancer cells but not normal cells suggests a conformational change of HSP70 in the lower pH environment characteristic of cancer cells (7). The presence of cell membrane embedded HSP70 has been found to increase the stability of cancer cells, thereby protecting tumors from environmental stress (8, 9).

Anti-HSP70 antibody, clone 1H11 (Catalog No. SMC-249) is unique from other commercially available antibodies in that it can bind to the extracellular region of the cell membrane embedded HSP70 protein, allowing researchers to differentiate between membrane bound and intracellular HSP70 across cancer cells types.

Further details are available here:
https://www.newmarketscientific.com/products?utf8=%E2%9C%93&simpleq=SMC-249

References:
1. Gribaldo, S. et al. (1999) J. Bacteriol. 181, 434-443. 2. Sharma, D. & Masison, D.C. (2009) Protein Pept. Lett. 16, 571-581. 3. Sharma, D. et al. (2009) PLoS. ONE.4, e6644. 4. Kampinga, H.H. & Craig, E.A. (2010) Nat. Rev. Mol. Cell Biol. 11, 579-592. 5. Nickel, W. & Seedorf, M. (2008) Annu. Rev. Cell Dev. Biol. 24, 287-308. 6. Multhoff, G. & Hightower, L.E. (1996) Cell Stress. Chaperones. 1, 167-176. 7. De Maio, A. (2011) Cell Stress. Chaperones. 16, 235-249. 8. Horvath, I. & Vigh, L. (2010) Nature. 463, 436-438. 9. Horvath, I., Multhoff, G., Sonnleitner, A., & Vigh, L. (2008) Biochim. Biophys. Acta 1778, 1653-1664.