Antibody News

1. Know your cytometer

The configuration of flow cytometers including light sources (lasers) and optics (mirrors, filters, and detectors) vary and a few, such as the Propel ZE5 Cell Analyzer, can detect up to 30 parameters in thousands to millions of cells. Lasers excite fluorochromes at fixed wavelengths and the emitted fluorescence is captured by optical detectors called photomultiplier tubes (PMTs). Setting PMT voltages correctly is important because a low setting can compromise signal detection. Furthermore, some instruments have interchangeable/swappable filters offering more flexibility when designing flow cytometry experiments.

2. Optimize antibody selections

To prevent complications during your experiment, take time to choose your antibodies and optimize their working conditions. Best practices dictate lowly expressed proteins or those with rare epitopes should be paired with antibodies conjugated to...

Microglia are resident macrophages in the central nervous system (CNS) that play roles in immune defense, inflammatory response, neurodegenerative disease and development. Identification of microglia has confounded researchers aiming to understand their biological function in the CNS, as they are molecularly and morphologically similar to other myeloid cells. For example, morphologically, microglia are indistinguishable from infiltrating blood derived macrophages.¹ Additionally, microglia are commonly purified for study based on their expression of molecular markers, such as CD11b^High and CD45^Low; although, these may change in disease states.^2,3

To fully understand and uncover the roles of microglia in the CNS, specific markers that accurately identify these cells are needed. Recently, investigators have embarked in efforts to elucidate specific...

Autophagy more than a cytosolic event

Autophagy is a cellular process whereby cytosolic components are broken down and eliminated or recycled. As a homeostatic mechanism, basal autophagic activity eliminates excess or abnormal proteins and organelles1. As an induced process, autophagy may be triggered by various external challenges, such as decreased nutrient and energy resources, and oxidative stress¹.

During autophagy, several cytosolic ATG (autophagy-related) and ATG-associated proteins drive the formation of the engulfing organelle^1,2. ATGs play key roles in the formation of the initial membrane vesicle or phagophore, and its subsequent elongation leading to the engulfment of cellular components. The resulting autophagosome fuses with lysosomes allowing the degradation of the sequestered content¹.

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With the wide variety of experimental techniques relying on primary antibodies, it is important to use both positive- and negative-controls in your antibody applications. We are generally more familiar with positive controls, which confirm antibody reactivity with a known-positive sample. However, we are often less familiar with adequate negative controls. An example of a negative control is an isotype control, which helps to confirm the specificity of a primary antibody.

Isotype controls may serve as a negative control for flow cytometry, immunohistochemistry and western blotting experiments. This control provides a measure of non-specific binding and may strengthen your experimental findings.

The antibody isotype or class denotes differences in the immunoglobulin’s heavy chain. When choosing an isotype control, select one that matches the clonality and...

The Western Blot – a tried and true experimental protocol where protein structures are separated via molecular weight/charge and transferred to a membrane before visualization by a chemiluminescent solution (say that three times fast!). Seems simple, right?  While the step-by-step process of a western blot has for the most part remained the same over the years, variations in solutions, procedures and reagents may increase the efficacy of your results. For example, when it comes to choosing a membrane for protein transfer there are good arguments for choosing between a PVDF and Nitrocellulose. Which one suits your protein sample best? 

Different types of cell death have classically been identified by discrete morphological changes. The hallmarks of apoptosis include cell shrinkage, nuclear fragmentation and membrane blebbing whereas necroptosis is characterized by cell swelling and plasma membrane breakdown. While these two forms of cell death are clearly distinct, substantial crosstalk occurs between them.  Accordingly, it is becoming increasingly important to understand how these processes differ and to understand ways to differentiate them in cellular populations. 

Apoptosis is a genetically programmed mechanism of cell death that is activated in response to cell stress, infection or developmental cues.   Apoptosis is split into two main pathways, the intrinsic and extrinsic pathways, and can be triggered by granzymes from natural killer (NK) cells and cytotoxic T lymphocytes.  The intrinsic pathway is regulated by the ...

The immune system is composed of a portion of T cells that express an invariant T cell receptor (TCR) alpha chain known as V alpha 24 J alpha 18. These highly conserved populations are referred to as iNKT populations and have the ability to rapidly produce cytokines following activation, making them hot targets for therapeutic research initiatives. In this article, we will review van derVilet et al’s study of the effects of administering high dose interleukin-2 to immunoregulatory cell subsets in patients presenting advanced melanoma and renal cell cancer.  This group uses antibodies in a variety of applications to determine whether these cells can rescue skin and kidney malignancies.  In this paper, dendritic cell subsets (DC), CD1d-reactive invariant natural killer T cells (iNKT) and CD4+CD25+ regulatory-type T cells are exposed to high-dose IL-2 therapy. 

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The CRISPR-Cas9 genome-engineering tool is a powerful opportunity for researchers to study individual gene function. CRISPR-Cas9, abbreviated for Clustered Regularly Interspaced Short Palindromic Repeats, is a bacterial defense system that can be reprogrammed to target specific areas of DNA followed by precise editing.  Essentially, CRISPR sequences are transcribed into short RNA sequences that will match the desired DNA sequence of interest. From here, the DNA is bound and cut, turning off its function.  While this recent discovery opened up doors for gene-targeted therapies, the mechanisms in which DNA cutting was achieved are somewhat debated. Simply put, when DNA is damaged, it sets out to repair itself, resulting in randomized nucleic acid insertions that are not needed. 

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Why: To identify a specific organelle or another cellular structure and to mark individual cells, it is necessary to counterstain them in immunocytochemistry/immunofluorescence (ICC/IF) assays.

How: Counterstaining is often performed with dyes or antibodies specific to the organelle or cellular structure of interest. For example, the nuclear counterstaining is carried out by using DNA helix intercalating dyes such as DAPI and Hoechst which can penetrate the cells and nuclei without permeabilization. Similarly, fluorescently-labeled phalloidin is used for counterstaining the cytoskeletal actin filaments and fluorescently-tagged wheat germ agglutinin (WGA) is employed for counterstaining the plasma membranes.

Important Considerations: When choosing counterstaining options in...

Novus employees will be participating in the March for Science alongside a global network of scientists to support the education and public importance of this topic for all.

We believe in the integrity of science and all that it holds for the future. Everyone is affected by it, and we believe in sharing this information with the public, outside of labs and journals.

Join us on April 22, 2017 at the March in D.C. or at your local satellite event. #BioTechneMarches

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• Minneapolis-St. Paul, MN
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• Washington, DC
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And more!

Follow our employees on social media with #BioTechneMarches throughout the weekend for scenes from around the world.

A tissue microarray is a fairly recent high-throughput application that allows researchers to test hundreds of tissue samples with antibodies of their choice at once.  Essentially, a tissue microarray is a paraffin block that is produced by a composition of tissue cores from paraffin donor blocks within defined coordinates to account for a variety of tissue types. Due to the success of the traditional IHC experimental method in advancing clinical research and drug discovery, the introduction of high-throughput IHC is pivotal to understanding the transformation of tissues from healthy to malignant.  This article will go deeper into the pros and cons of tissue microarray, as well as introduce tissue array sets available at Novus Biologicals and real world applications. 

Apoptosis, or programmed cell death, is controlled by a caspase signal cascade that activates downstream signals to induce the morphological changes used to differentiate apoptosis from other forms of cell death.  Novus Biologicals offers a variety of antibodies and tools to detect the different morphological indicators of cell death. 

Nuclear Fragmentation

Nuclear condensation starts at the nuclear membrane, hallmarked by the formation of a ring-like structure.  Further into apoptosis, the nucleus full fragments in a process known as “karyorrhexis”.  Primary antibody markers that bind and recognize DNA such as ICAD (inhibitor of caspase-activated DNAase) and Lamin B can be used to visualize nuclei.  Furthermore, TUNEL, or terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick end labeling, is a popular method of apoptotic cell...