Exosome Purification via Size Exclusion Chromatography (SEC)
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SEC, also known as molecular sieve chromatography, is a chromatographic method in which molecules in
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Fluorescent dye probes or fluorophores are often described as fluorescent chemical sensors because they can absorb light at a given wavelength and emit light at a higher wavelength to produce fluorescence in various colors. Generally, they could be divided into organic dyes (e.g., fluorescein, rhodamine, AMCA), biological fluorophores (e.g., green fluorescent protein, phycoerythrin, allophycocyanin), and quantum dots. Due to the high sensitivity, technical flexibility, and quick response time of fluorescent dyes, they have become widely-used tools for quantitative detection and visual imaging.
Compositions of Fluorescence Probes
Biomolecules require to be marked by fluorescent labels before they can be observed through fluorescence microscopy. That's to say, researchers need to attach fluorescent dyes to organic compounds inside the molecules since fluorescent labels are sensitive even at low concentrations, durable for extended durations of time, and therefore do not affect the functionality of the protein targets.
A fluorescence probe consists of three necessary components, including a signaling unit (chromophore), a separator (chemical bridging), and an attaching unit (receptor), the alteration of which enables researchers to design and tailor probes for particular targets. Chromophores usually are organic pigments that can only capture visible light. Diverse auxochromes and functional groups that can change the ability of chromophores to collect light are often added to these organic pigments. Auxochromes can be added to alter the spectrum position and brightness of the generated color.
Specific sensors with red or green fluorescence, which are two of the most sought hues in the range of the electromagnetic spectrum, are widely used in biochemistry, immunofluorescence, immunostaining, and a series of processes. A symbolic application of fluorescent sensors is in the field of imaging living tissue, which can reduce cellular fluorescence intensity and specific binding in multicolored confocal microscopy.
Fluorescence Imaging Techniques to Visualize Target Protein
There are two techniques that can be used to visualize target proteins by taking advantage of fluorescence imaging. One is the aforementioned method that biologically attaches a fluorescent dye to a target molecule to form an intrinsic chromophore, and the other is using fluorescently tagged antibodies to selectively bind to a targeted protein. But a final test is usually required in practice, especially in cases where fluorescent proteins are not applicable, for example, in histology samples.
Advantages & Disadvantages of Fluorescent Proteins
The fact that fluorescent proteins are proteins themselves could be a further drawback given certain proteinous properties inside a cell. Specifically, it may lead to malfunction or incorrect interpretations when researchers investigate associated or particular proteins of interest. Moreover, if target antibodies are available, immunofluorescence would be more time-effective than luminescent proteins that require desired gene replication or DNA insertion into the target cell.
But it's undeniable that fluorescent proteins are useful gene function sensors in various applications as well as promising indicators to monitor specific cellular components, identify proteins in human tissues, and track enzyme dispersion and intracellular pathways. In addition, fluorescent proteins complexed with antibodies or other biochemicals are also important in immunocytochemistry, electron microscopy, Western blot analysis, immunohistochemistry, and fluorescence-based immunoassay assay (FLISA).
Development of New Fluorescence Probes
Novel fluorophores have enhanced the potential for the specific detection of enzymatic activities and their interactions. And analyses of cellular procedures and systems, which were previously unachievable, are now accessible due to improved fluorescent dyes. What's more, fluorescent dyes have various benefits including high specificity, high sensitivity, as well as the capacity to fine-tune optical characteristics, such as lifetime, emitted and activation spectra, brightness, and anisotropy. And they may be used in model organism research, including such tissues and cells.
Therefore, it's necessary to design new fluorescence probes with improved characteristics, functions, and structures.
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March 7, 2022
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RNA is a polymeric molecule composed of nucleotides that are required for gene coding, decoding, regulation, and expression. In molecular biology, DNA's genetic information gets transcribed into multiple copies of messenger RNA (mRNA), and then mRNA is translated into proteins. This is why cells can synthesize several protein molecules from a single gene at a given point of time. However, only 1-4% of total RNA are mRNA, the rest of which are generally non-coding, called non-coding RNA (ncRNA). ncRNA is unable to be translated into protein but there are many types of them, which have different biological functions.
Therefore, it's necessary to analyze the total RNA molecules for a better understanding of gene expression, which is called transcriptome analysis. Gene expression analysis can be used to monitor the response of a gene to treatment with a compound or drug of interest. Leveraging features of the next-generation sequencing (NGS) https://www.creative-biolabs.com/suprecision/, RNA sequencing (RNA-Seq) can efficiently detect and quantify RNA in a biological sample at a given point in time. Researchers often take advantage of the high-throughput sequencing data, broad dynamic range, and high sensitivity from NGS to investigate the whole transcriptome of a single cell or a population of cells without using probes and to detect differentially expressed genes.
Protocol of RNA-seq
Though with a slight difference for different RNA sequencing experiments, the protocol is generally similar and involves these steps:
The complete set of transcripts of the target cell or tissue should be isolated from the sample.
If a specific type of RNA is to be profiled, researchers need to purify and enrich that kind of RNA from the total RNA molecules.
Make an RNA sequencing library that consists of a pool of complementary DNA (cDNA) fragments with adapters attached.
Perform sequence with the next-generation sequencing platform.
Finally analyze the short-read sequencing result.
Before being sequenced by the next-generation sequencing platform, the RNA sample is required to be fragmented, end-repaired, and collected into adapter-ligated libraries. The RNA-Seq library preparation https://www.creative-biolabs.com/suprecision/category-rna-library-prep-284.htm is dependent on the NGS platform applied and can influence the results generated by NGS experiments. Fortunately, most service providers will offer library preparation kits for directional and non-directional RNA library prep that match their platforms for sequencing with stable reagents and streamlined workflows.
Preparation of RNA Sequencing Library for NGS
There are two common library preparation methods, ligation-based library prep and tagmentation-based library prep. The ligation method separates the steps of DNA fragmentation and ligation of adapters to the ends of the fragments, while the tagmentation method combines DNA fragmentation and adapter ligation into one reaction step. Take ligation-based library preparation as an example, here are the two major steps required to prepare an RNA sequencing library.
Addition of adapters
A ligase enzyme covalently will link the adapter and insert cDNA fragments, making a complete library molecule. These adapters have different functions so their sequences specific to the NGS platform should also be attached to the flow cell to allow RNA sequencing.
PCR amplification
Amplification of libraries is optional and depends on the adapter type and input used. The remaining oligonucleotides and small fragments should be removed using magnetic beads or a spin column if PCR amplification is applied.
Besides, researchers might need to consider reverse transcription of RNA and cDNA synthesis and whether or not to preserve strandedness. The adaptor sequences that will be specific to the sequencing platform must also be added to the end of the fragments. Once the RNA sequencing library gets prepared, researchers can go ahead with the next step in the next-generation sequencing workflow.
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Bacteria are covered by polysaccharides at the outer surface in the form of capsules, glycoproteins, or glycolipids. Such a bacterial sugar coat constitutes the principal antigens in most pathogenic bacteria and plays an important role in host-pathogen interactions. With more understanding of polysaccharide biosynthesis and its interplay with polymer modification and synthesis, scientists have recognized the research potential of polysaccharides in developing novel antibacterial strategies and novel applications like epidemiological markers.
Bacterial polysaccharides mainly are carbohydrates like capsular polysaccharides (CPS) and lipopolysaccharides (LPS). Capsular polysaccharides are highly-hydrated homo- or hetero-polymers that are composed of repeating sugar units joined by glycosidic linkages. They usually are inserted into the cell surface of bacteria by covalent attachments to either phospholipid or lipid-A molecules. Lipopolysaccharide, on the contrary, is a membrane component characteristic of Gram-negative bacteria consisting of lipid A, core-oligosaccharide, and O-polysaccharide (or O-antigen) joined by a covalent bond. With a series of advanced technologies, including chemical degradation techniques, nuclear magnetic resonance spectroscopy, and mass spectrometry techniques, researchers now can comprehensively carry out structural characterization and analysis of bacterial polysaccharides and understand their functions.
CPS usually constitutes the outermost layer of the cell and gets involved in mediating interactions between bacteria and the environment, for which polysaccharide capsules are implied as important virulence factors for several bacterial pathogens. Moreover, CPS can protect bacteria from phagocytosis if the pathogen is attacked by innate immune responses. Capsular polysaccharides prevent the activation of phagocytosis by decreasing antibody opsonization and masking ligands for phagocytic cell attachment. Therefore, polysaccharides antibodies as biomarkers(https://www.creative-biolabs.com/anti-glycan-antibodies/anti-glycan-antibody-applications.htm) have become the research hotspot in the field of disease diagnosis and treatment. Researchers at the Queensland University of Technology have successfully characterized the genomic loci in Acinetobacter baumannii that are responsible for cell-surface polysaccharide synthesis and have proven them to be effective epidemiological markers to track A. baumannii.
Lipopolysaccharides are large molecules localized in the outer layer of the bacterial membrane and populate much of the cell surface. LPS can establish a permeability barrier, protecting bacteria from toxic molecules such as antibiotics and bile salts. In addition to being a key component of the cell envelope, LPS also contributes to host-pathogen interactions with the innate immune system. Bacterial adaptive changes, including modulation of LPS synthesis during chronic infection, could protect disease by preventing phagocytosis and adhesion to epithelial cells with O-antigen lipopolysaccharide or enhancing host immune response evasion with a production of less immunogenic lipid A, etc. In some cases, the immune response against the bacterial polysaccharide could be too dramatic to be toxic to the host, which confers protection against the disease in some depth.
Polysaccharides could also be found in fungi and yeast in the form of glucans, chitin, and mannans, playing a major role in the cell wall structure. Current research on fungal polysaccharides (https://www.creative-biolabs.com/anti-glycan-antibodies/polysaccharide-other.htmz) mainly aims at identifying influential factors for their biological activity and elucidating their interactive role in various chemical medicine. The biological activities of fungal polysaccharides are shown to influence anti-tumor, anti-microbial, immune-stimulation or immunomodulatory activity, nutritional component, and hypoglycemic activity.
With more understanding of the structure of various polysaccharides, details about the mechanism of action of polysaccharides in different systems are being revealed. Polysaccharides of bacteria and fungi will share more extensive applications in diagnostics and therapeutics.
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The complement system maintains tissue homeostasis and integrity, which initiates and coordinates immune reactions attacking and clearing tagged foreign cells and altered self cells, such as apoptotic particles. However, the activation of complement generates highly toxic products. And consequently, it needs to be precisely and tightly regulated by multiple inhibitors and regulators, including integral membrane proteins, surface-bound regulators, and soluble effectors, which are distributed in the body fluids and plasma.
Background
As dysregulation of the balance between complement activation products and regulators results in different autoimmune diseases, diverse complement regulators are considered as the natural modifiers, effectively preventing the complement system from attacking the host cells.
Research in early complement-directed drug discovery has further demonstrated that complement regulators, especially soluble complement regulators, are important therapeutic targets for autoimmune diseases.
About Soluble Complement Regulators
The first breakthrough was reached in complement-directed drug discovery with the expression of a soluble form of complement receptor 1 (sCR1). It's a molecule featured with both decay accelerator and cofactor activity, additionally showing a potential to inhibit the classical and alternative pathways.
sCR1 exhibited inspiring results in treating I/R injury and several other diseases in experimental animal models. These promising results facilitated the therapeutic drug, TP10, which is widely used after coronary artery bypass graft surgery. A large placebo-controlled phase 2 trial with 564 high-risk patients undergoing cardiac surgery verified the efficacy of TP10 complement. The complement activation was inhibited for up to 3 days by a single intravenous bolus of TP10 immediately before surgery.
The soluble forms of complement regulators have multiple advantages over anti-cytokine antibodies, such as high specificity, high affinity, and less possibility to induce immune responses and attenuate the actions of agents.
Common Soluble Complement Regulators
The increasing recognition of the involvement of complement regulators in health and disease has prompted the development of complement-directed drugs. And a variety of creative therapeutic approaches and products are emerging.
sCR1/TP10 (Avant Immunotherapeutics)
As aforementioned, the first advance in soluble complement regulators was the expression of sCR1. It has been the most extensively developed regulator of complement activation for a lot of experimental animal disease models, including injury, arthritis, glomerulonephritis, myasthenia gravis, allergic encephalomyelitis, acute myocardial infarction, lung transplantation, and post-cardiopulmonary bypass syndrome.
CAB-2/MLN-2222 (Millenium Pharmaceuticals)
Soluble forms of MCP, DAF, and CD59 have also been recognized as therapeutics with the development of different approaches for the clinical substitution and inhibition/modulation of complements. Though DAF and MCP each only offer a single regulatory activity, a recombinant chimera of their extracellular parts has been developed with the result of the sDAF-sMCP hybrid, which was initially named "complement activity blocker 2" (CAB-2) and later clinical trials renamed it as MLN-2222.
Recent research indicates that recombinant CD59 may be a promising therapy for paroxysmal nocturnal hemoglobinuria (PNH) as it's related to the decreased expression of membrane-anchored proteins, including CD59 and DAF, on erythrocytes.
Other related regulators include soluble complement inhibitors C4-binding protein (C4BP), factor H, and factor I complement.
C4BP and factor I are responsible for the regulation of C4b, which shows promising effects on the treatment of diseases associated with abnormal or loss of complement control. Factor H performs a deactivator in the complement system. Moreover, C4BP proves to inhibit the classical pathway while factor H inhibits the alternate pathway, which may suppress self cell damages and inflammation.
In brief, complement activation takes an essential part of the mechanism of pathogenesis of human diseases, and soluble complement regulators are encouraging therapeutic targets to suppress or prevent complement-mediated diseases. However, researchers need more experiments based on optimized and validated protocols to develop more effective and safer anti-complement therapeutics, in which the assistance from soluble complement regulator development services will make success with half the effort.
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The prominent manufacturer with considerable experience in antibody production and engineering, Creative Biolabs, catches the technology of antibody array and makes it more efficient and accurate by altering and customizing on-chip performances, specificity, antibodies renewability, scaling-up, and molecular design.
The antibody array (also known as antibody microarray) is a specific form of protein microarray using high-throughput techniques. In modern biotechnology, it turns out to be an important protein expression profiling tool and is widely applied to analyze certain cytokines, chemokines, and growth factors of diseases.
Qualified Antibody Arrays Aid Biology-driven Studies & Clinical Research
The technique of antibody array is acknowledged as it measures many proteins simultaneously in a single experiment. Compared with traditional single analysis methods, properly using antibody array technology takes less time, effort, and costs in protein expression profiling.
Those obvious advantages increase the use of antibody arrays in different fields and also promote many antibody array products in the market. Antibody array products and technology should improve modern biology studies, clinical research, and it also helps detect cancer and inflammatory indications.
Five Crucial Points for An Accurate Recombinant Antibody Array
Expertise team at Creative Biolabs thoroughly comprehend the antibody array technology and deeply engage in the core principles of antibody array. All operations are based on the accurate recognition ability between an antibody and the corresponding target antigen.
The antibody array technology requires that antibodies are spotted onto a solid support, like a glass slide, and then incubated with biological samples. Later, the activity between the antibody and its specific antigen is detected, scanned, and analyzed using professional methods.
Creative Biolabs holds that “when designing and developing custom antibody microarray services, 5 important factors should be taken into consideration. And it’s vitally important to select the best antibody design that matches the specific requirements of a certain application.”
On-chip performances
Range of specificity
Renewability
Scaling-up
Molecular design
On the condition of these crucial factors, Creative Biolabs levels up the accuracy, simplicity, and efficiency of antibody array.
Antibody Array Products and Customized Services
In addition to providing antibody array technology platforms and various antibody array products, Creative Biolabs is also skilled at custom antibody array services and products when extra and specific applications are needed.
For instance, several different antibody array technologies have been developed, including sandwich-based arrays and label-based arrays.
Meanwhile, Creative Biolabs yields prolific platforms and products, including membrane antibody arrays that are easy to handle and severely cost-effective, glass slide antibody arrays that waste less volume of a sample and more suitable for high-throughput liquid handling systems, and 96-well plate-based antibody arrays that have a high-throughput capability.
More information about antibody arrays can be reached at https://www.antibody-creativebiolabs.com
About Creative Biolabs
Creative Biolabs is a prime life-science company, having engaged in antibody production and engineering for years. Based on antibodies for more than 1000 human, mouse, rat and other species targets, a series of antibody array products and customization services are available in Creative Biolabs’s recombinant antibody array technology platforms.
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