Antibody News

PINK1 as a Mitochondrial Health Sensor and Neuroprotector

Tuesday, September 19, 2017 - 09:40

By Bethany Veo, PhD

Mitochondria are most commonly known as the power houses of the cell, facilitating major functions such as oxidative phosphorylation and cellular respiration.  Maintenance of mitochondria is essential to a cells' physiological homeostasis and requires oversight by several factors.  PINK1 is a serine/threonine protein kinase which localizes to the mitochondrion and regulates its function and turnover by sensing when mitochondria are damaged.1  The foremost mechanisms of mitochondrial health upkeep include fusion and fission, mitophagy, and mitochondrial transport. PINK1 is critical for mitochondrial health by facilitating all of these pathways, which serve as a quality control system to remove dysfunctional or damaged mitochondrion from the cell.  In fact, mutations in PINK1 are linked with Parkinson's disease, where dysfunction in mitophagy, mitochondrial clearance and...

Autophagy: Pro or Anti-tumorigenic? And the role of epigenetics in this debate

Monday, September 11, 2017 - 11:26

By Christina Towers, PhD

Autophagy is an evolutionarily conserved process that cells use to break down damaged cytoplasmic constituents in order to fuel cellular metabolism, particularly in instances of stress. This process has been heavily implicated in a variety of diseases, most noteworthy are neurological disorders and cancer. The role of autophagy in cancer is context dependent and somewhat controversial1. It was originally suggested by Dr. Beth Levine's group that autophagy is tumor suppressive, a claim supported by loss of the core autophagy gene, BECN1, in many tumor types including breast, prostrate, and ovarian2. However, critics of this notion point out the important observation that BECN1 is adjacent to the heavily deleted tumor suppressor gene, BRCA1,...

The Proteasome and Autophagy Pathways in Alzheimer's Disease

Tuesday, September 5, 2017 - 09:42

The neurodegenerative disorder, Alzheimer's disease, is responsible for 60 to 80% of all dementia cases.1   Neurodegeneration occurs in response to the accumulation of amyloid-β plaques and neurofibrillary tangles composed of hyperphosphorylated tau. The proteolytic processing of AβPP (amyloid β precursor protein) by β-secretase and γ-secretase releases Aβ fragments of 40 and 42 amino acid residues which miss-fold and aggregate into the pathogenic plaques.2  Similarly, proteolytic processing of phosphorylated tau releases monomers that are targeted to the 26S proteasome for degradation. However, proteasomal efficiency is poor when it comes to degrading ubiquitinated tau as these substrates lead to more neurofibrillary tangle aggregates.2 The inability to clear these accumulating plaques intracellularly results in a significant amount of oxidative and cellular stress leading to progressive neuronal loss and...

Application guide: Methods to monitor Autophagy

Tuesday, August 29, 2017 - 15:50

Autophagy is an essential process that cells utilize to degrade and recycle damaged material and fuel metabolism, especially under stress.  The process is evolutionarily conserved and complex, relying on over 20 key proteins. Induction of autophagy is mediated by the formation of the

Novel Insights into Hypoxia Induced AKT Signaling

Thursday, August 24, 2017 - 10:51

Hypoxia is a common feature of most tumors and is a product of rapid cell growth and poor vascularization1. When oxygen availability is low in the tumor environment, the hypoxia inducing transcription factors (HIFs) regulate a variety of signaling programs that can affect the balance between tumor cell apoptosis2 and autophagy3.  In normoxia, HIFs are bound by the von Hippel-Lindau protein (VHL) in the cytosol where it is degraded by the proteasome, however, under hypoxia HIFs are translocated to the nucleus where they activate survival signals. Additionally, HIF mediated signaling can increase the metastatic capabilities of tumor cells and facilitate the pro-metastatic phenotypes of epithelial to mesenchymal transition (EMT), alter tumor cell metabolism, increase vascularization and angiogenesis, as well as suppress immune reactivity1. Together these HIF regulated pathways help cancer cells to thrive and activate pro-...

pSIVA a Biological Switch and Sensor of Apoptosis

Monday, August 21, 2017 - 09:27

Altered cellular membrane integrity is one of the earliest signs of apoptosis.1,2 One key change during this event is the movement of phosphatidylserine (PS) from the inner leaflet of the cell membrane towards the cell surface. This process, due to the inactivation of flippase- and activation of scramblase-enzymes, is inducible, reversible and dependent upon calcium release from the endoplasmic reticulum.1 Exposure of PS acts as an "eat me" signal, prompting phagocytosis of apoptotic cells.3 In the absence of PS exposure during apoptosis, dying cells would escape the immune system ensuing an inflammatory response.2 Thus, PS externalization has emerged as a useful event and target for monitoring the progression of apoptosis in real-time.

The discovery of a natural ligand, Annexin A5, which binds reversibly, selectively and with high-affinity to exposed PS prompted its development as a biological tool for the...

Applications Focus: 5 tips for Flow Cytometry Panel Design

Monday, August 14, 2017 - 13:14

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...

Article Review: Glucose-induced transcriptional regulation in cancer

Tuesday, August 8, 2017 - 14:20

Epigenetic mechanisms have been implicated in many physiological and pathophysiological processes. Among these, histone modifications including methylation, phosphorylation, acetylation and ubiquitination, significantly modify gene expression. In cancer, specific abnormal epigenetic changes are thought to impart tumor cells with properties that facilitate their survival.1 Recently, a new study uncovered a novel mechanism regulating epigenetic changes of importance in cancer. Briefly, a sequence of post-translational modifications triggered by changes in glucose levels were identified to positively impact tumor sphere formation and tumor engraftment.2

Histone acetylation is generally associated with an increase in gene transcription.3 Formation of this epigenetic mark is catalyzed by...

Application Focus: New targets for immunostaining analysis of microglia

Tuesday, August 1, 2017 - 09:35

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 CD11bHigh and CD45Low; 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...

Apoptosis and Necroptosis Part II: Inhibitors of apoptosis proteins (IAPs); Key regulators of the balance between necroptosis, apoptosis and survival

Tuesday, July 25, 2017 - 09:53

In the first installment of this two-part blog post titled "Apoptosis and Necroptosis: Important factors to identify both types of programmed cell death", the mechanisms by which cell death occurs and ways to identify these pathways were discussed. In this next segment, we focus on the molecular factors regulating the choice between programmed cell death and survival signaling.

Inhibitors of apoptosis proteins (IAPs) regulate cell death and survival signaling via different mechanisms. In mammals, a total of eight IAP family members have been identified1. Among these, X-linked IAP (XIAP), and cellular IAP1 and 2 (cIAP1 and cIAP2) suppress programmed cell death signaling...

Epigenetic mechanisms: new insights on the regulation of autophagy

Tuesday, July 18, 2017 - 08:17

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 organelle1,2. 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.

...

Applications Focus: Labeling with multiple secondary antibodies

Wednesday, July 12, 2017 - 09:15

Multiple fluorescent labeling with secondary antibodies for immunocytochemistry and immunohistochemistry is a powerful tool to examine the behavior and interactions of more than one protein in a cell or tissue sample.  However, there are a few guidelines to follow to make sure your samples are correctly labeled. Read our top five tips for a successful multiple antibody labeling experiment:

  1. Select primary antibodies raised in different host species. If you must use antibodies from the same host, use different IgG isotypes. Perform single staining of the antibodies prior to introducing multiple antibodies and secondary antibodies to the experiment, this will allow you to understand their isolated behavior and expression.

  2. When selecting secondary antibodies, make sure they come from the same host species, but have different fluorophores within a safe range of spectra to avoid overlap. It is a...

Taking a closer look at isotype controls in antibody applications

Thursday, June 29, 2017 - 14:32

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...

Applications Guide: How to choose fluorophore combinations for Flow Cytometry

Wednesday, June 14, 2017 - 11:22

Flow cytometry is an experimental method that was developed to label and examine a high volume of cells in an extremely rapid rate using antibodies conjugated to fluorophores.  The basic concept of flow cytometry is that a cell suspension is situated into a single stream, which then passes through a light source that uses detectors to generate data sets based off cellular properties.  More specifically, the fluorescent light emitted by fluorophores conjugated to antibodies is channeled through selected filters to sort based off preset parameters or targets used. Flow cytometry is particularly useful in cell viability and proliferation assays, as well as diagnosing disease (particularly blood cancers).  When creating a panel of fluorophores for your flow cytometry panel, it is important to follow a few basic guidelines.

First, it is important to understand your flow cytometer.  You should know the type of laser in your instrument, the number of lasers present and...

Make the most of your membrane: PVDF vs. Nitrocellulose

Wednesday, June 7, 2017 - 11:38

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? 

...

 

PVDF

Nitrocellulose

The role of STING/TMEM173 in gamma and encephalitis Herpes Simplex Virus (HSV)

Wednesday, May 31, 2017 - 10:57

Stimulator of interferon genes (STING), also known as TMEM173, promotes the production of the interferon’s IFN-alpha and IFN-beta.  STING possesses three functional domains: a cytoplasmic C-terminal tail, a central globular domain, and four N-terminal transmembrane motifs that attach it to the ER.  The role of STING in the immune response is specific to its ability to sense nucleic acids, particularly dsDNA.  When STING is over induced, the protein IRF3 undergoes a nuclear translocation resulting in IFN induction, which in turn activates the innate immune response. The herpes virus is a DNA based virus targeted to DNA-sensing pathways that has the capacity to elicit latent recurrent infections on a host. Because of the overlap between the herpes virus affecting DNA-sensing and the ability of STING to sense nucleic acids, using...

Apoptosis and Necroptosis Part I: Important factors to identify both types of programmed cell death

Friday, May 26, 2017 - 11:22

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 ...

Rules for Selection of Fluorochromes in Multicolor ICC/IF

Monday, May 22, 2017 - 10:24

Immunocytochemistry/immunofluorescence (ICC/IF) involves visualization of antigens in cultured or smeared cells with the use of fluorochrome-labeled antibodies. Various combinations of fluorochrome-labeled antibodies make it possible to simultaneously detect multiple antigens in the same sample. However, every experiment involving multiplex or multicolor ICC/IF requires selection of an optimal set of fluorochromes. Poor selection of fluorochrome combinations can make it difficult to distinguish individual antigens, especially when the targets under consideration are co-localized at the sub-cellular level. Here we have outlined key rules for selecting fluorochromes to minimize potential errors and to simplify the selection process.

1. Check for fluorochrome(s) compatibility with your microscope’s filter sets

Use spectra viewers and fluorochrome reference charts to determine the maximum excitation (Ex) and emission (Em) wavelengths...

Article Review: Effects of the administration of high-dose interleukin-2 on immunoregulatory cell subsets in patients with advanced melanoma and renal cell cancer

Friday, May 19, 2017 - 10:38

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. 

...

Article Review: Dual effects of carbon monoxide on pericytes and neurogenesis in traumatic brain injury

Tuesday, May 9, 2017 - 08:58

Traumatic brain injury (TBI) currently contributes to nearly 30% of all injury deaths in the United States.  Characterized by an abrasive head injury that interrupts normal brain function, TBI can range from mild to severe.  Mild symptoms can present themselves as excessive tiredness, difficulty concentrating and lack of clear thinking.  Severe cases of TBI are hallmarked by unusual behavior, seizures and loss of consciousness.  Research has shown that on a molecular level TBI triggers various mechanisms of cell death alongside attempted tissue recovery, therefore Choi et al sought out to test the treatment of carbon monoxide on rescuing the effects of TBI.  Their article titled “Dual effects of carbon monoxide on pericytes and neurogenesis in traumatic brain injury” tests the effects of CO-releasing molecular 3 (CORM-3) and N-tert-butyl-a-phenylnitrone (PBN) on a mouse model of controlled cortical impact TBI.

...

Recent advances in CRISPR-Cas9

Thursday, May 4, 2017 - 09:49

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. 

...

Pathway Highlight: Three key factors that contribute to cellular heterogeneity in apoptosis

Monday, May 1, 2017 - 08:54

Have you ever wondered why cells in the same population respond differently to an apoptotic stimulus? Apoptosis, a form of programmed cell death, is vital for the removal of unwanted or damaged cells. As with most cellular processes, too much or too little activation can be detrimental and lead to various diseases including autoimmune disorders and cancer. While this process is tightly regulated, cells undergo apoptosis in a non-synchronized manner, which complicates the analysis of apoptotic events.  Here are 3 keys factors that determine how an individual cell will respond to an apoptotic stimulus.

caspase-3 antibody

Processed caspase 3 was detected in immersion fixed anti-FAS treated Jurkat human acute T cell leukemia cell line using Human/Mouse Active Caspase 3 Polyclonal Antibody (...

Why do counterstaining in ICC/IF and how?

Friday, April 28, 2017 - 10:26

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...

The role of MHC Class II RT1B and immune response post brain injury

Tuesday, April 25, 2017 - 09:39

The major histocompatibility complex (MHC) is responsible for binding peptide fragments arising from pathogens in order to display them on the cell surface for recognition from immune cells.  Once recognized, the foreign pathogen is typically evaded. The MHC complex is broken into two categories, MHC Class I proteins and MHC Class II proteins.  MHC complex I and II proteins are all very different and contain specific molecules to bind different peptides – in fact, they have been described as the most polymorphic genes there are. The MHC Class II RT1B antibody can be used to bind the monomorphic determinant of the rat I-A antigen, which is found on B-lymphocytes, dendritic cells and some macrophages. Often times, a neuroinflammatory response develops after brain injury and remains for weeks post injury.  Using a MHC Class II RT1B antibody is a useful way to understand...

Novus Marches for Science!

Friday, April 21, 2017 - 10:15

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

We will be marching in our Bio-Techne communities:

  • Minneapolis-St. Paul, MN
  • Denver, CO
  • San Jose, CA
  • Washington, DC
  • Boston, MA
  • Bistol, UK

And more!

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

Pages


Blog Topics


Archives