Antibody News

How to switch from apoptotic to necroptotic cell death? Answer: Autophagy!

Tuesday, November 21, 2017 - 08:49

By Christina Towers, PhD.

It has long been known that programmed cell death can take place in many forms.  The most well characterized form, apoptosis, occurs when either extrinsic or intrinsic stimuli stimulate signaling cascades that result in a series of caspase cleavage events resulting in cleavage and activation of the effector caspase-3.  The end result is cell shrinkage, condensation and cleavage of chromosomal DNA, and plasma membrane blebbing that leaves the cell membrane intact1.  Apoptotic cells are engulfed by antigen presenting cells and induce anti-inflammatory and tolerogenic immune responses2.  Alternatively, necroptosis, a programmed form of necrosis, is less characterized.  However, it is now known that this process is also tightly regulated and mediated by a complex...

Autophagy’s Paradoxical Role in ALS

Tuesday, November 14, 2017 - 10:21

By Yoskaly Lazo-Fernandez, PhD

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a progressive neurological disease that affects the motor neuron system and thus voluntary control of muscle movement. This disease belongs to a broader group of disorders known as motor neuron diseases, characterized by progressive degeneration and death of motor neurons. There are two types of motor neurons, including upper- and lower-motor neurons, based on the position of their somas within the CNS. Upper motor neuron somas are located in the motor cortex and their axons descend to the spinal cord where they activate the lower motor neurons. Lower motor neurons have somas within the spinal cord and extend their axons peripherally to innervate skeletal muscles. In ALS patients...

Autophagy as a Therapeutic Target: The Double-edged Sword

Tuesday, November 7, 2017 - 09:46

By Christina Towers, PhD

Autophagy is an important cellular process that facilitates the degradation of damaged cytoplasmic material and toxic protein aggregates. Its role in neuronal function is apparent by the neurodegenerative phenotypes observed in autophagy deficient genetic mouse models. Mice with neuron-specific knock out of the core autophagy protein, ATG7, are viable but most go on to develop behavioral defects and eventually massive neuronal loss in the cerebral corticies1.

But perhaps the most causative links between autophagy and neurological functioning have been observed in animal models of progressive neurodegenerative diseases including Alzheimer’s, Parkinson’s, and Huntington’s disease. Each of these devastating diseases is characterized by a build-up of toxic protein aggregates: β-...

Polyglutamine Tracts as Autophagy Regulators

Tuesday, October 31, 2017 - 09:20

By Yoskaly Lazo-Fernandez, PhD

Polyglutamine tracts (polyQ tracts) are long chains of glutamine amino acidspresent in the sequence of many proteins. The length of polyQ tracts within proteins varies significantly as normal alleles of specific genes usually contain different number of the cytosine-adenine-guanine (CAG) nucleotide repeats1.

It has long been known that polyQ tracts play a role in the etiology of several inheritable neurodegenerative disorders, including spinocerebellar ataxia, and Huntington's disease2. These 'polyglutamine diseases' result from the excessive elongation of a polyQ tract in a particular gene which causes the resultant protein to become toxic. The toxicity of mutated polyQ tracts has been extensively studied and several explanatory hypotheses have been proposed: aggregation of polyQ tract proteins, transcriptional dysregulation, mitochondrial dysfunction, and impairment of both the ubiquitin-...

Autophagy and Apoptosis: who regulates whom?

Monday, October 23, 2017 - 13:13

By Christina Towers, PhD

Apoptosis, or programmed cell death, is the result of an intricate cascade of signaling events that is initiated by extrinsic (death receptor mediated) and intrinsic (mitochondrial mediated) stimuli.  Extrinsic apoptosis is initiated by ligand binding to death receptors followed by signaling cascades that activate caspase-8, also known as the initiator caspase.  The effector caspase-3 can be activated by caspase-8, but is usually activated downstream of the intrinsic pathway, and often the extrinsic pathway will converge on the intrinsic for more efficient programmed cell death. Intrinsic signaling takes place on the mitochondrial membrane, where BH-3 only proteins activate mitochondrial outer membrane permeabilization (MOMP) and the release of mitochondrial proteins like cytochrome c.  Subsequently, the apoptosome is formed by a...

Beyond Genes: Treating Memory Loss with Minimum Adverse Effects

Tuesday, October 17, 2017 - 12:43

By Jamshed Arslan Pharm.D.

Alzheimer’s disease (AD) robs people of their memory and identity. One characteristic feature of AD is the increased expression of the enzyme, histone deacetylase-2 (HDAC-2). This protein stops the expression of some memory-forming genes by condensing them. So, memory can be enhanced by inhibiting HDAC-2. However, this approach is dangerous since HDAC-1, a close family member of HDAC-2, would also become inhibited, resulting in neuronal death and other toxic effects. New research,[1] led by Li-Huei Tsai at the Massachusetts Institute of Technology, Cambridge, suggests a novel way to specifically repress HDAC-2 by targeting the domain of HDAC-2 that makes a complex with the transcription factor, Sp3.

...

There's an autophagy for that!

Tuesday, October 10, 2017 - 13:09

By Christina Towers, PhD

A critical mechanism that cells use to generate nutrients and fuel metabolism is through a process called autophagy.  This process is complex and involves over 20 different proteins, most of which are highly conserved across species.  It involves the formation of a double membrane structure known as an autophagasome that fuses with the lysosome to facilitate the degradation of cytoplasmic material.  While bulk autophagy is thought to be largely non-specific, clearing damaged proteins from the cytoplasm, recent studies have begun to highlight more selective forms of autophagy.  Selective autophagy, also coined organellophagy, facilitates the degradation of specific organelles that are damaged or targeted for recycling.  Thus far, researchers have begun to investigate the selective degradation of mitochondria, peroxisomes, endoplasmic reticulum (ER), nuclei, and chloroplasts in plants, all mediated through variant forms of...

HIF-1 alpha and HIF-2 alpha in Muscle Development

Tuesday, October 3, 2017 - 11:42

By Bethany Veo, PhD

Our muscle cells develop from myogenic stem cells or satellite cells which have the capacity to grow, self-renew, differentiate, and regenerate under the influence of changing oxygen levels sensed from the microenvironment. Hypoxia-inducible factors HIF-1 alpha and HIF-2 alpha are transcription factors that respond to low oxygen levels, activating the expression of target genes. The current understanding of the role that HIF-1 alpha and HIF-2 alpha play in muscle development is contradictory. Case in point, upregulation and downregulation of HIF-1 alpha inhibit myoblast differentiation depending on the oxygen level in vitro.

Transgenic models including mice lacking HIF-2 alpha exhibit early embryonic arrest and HIF-1 alpha knockouts are lethal. Whereas HIF-1 alpha knockouts generated with a different Cre-...

Necroptosis in Health and Disease

Tuesday, September 26, 2017 - 09:22

By Bethany Veo, PhD

Necroptosis occurs when cells fail to undergo apoptosis following inflammatory, oxidative or ischemic stressors. Necroptotic cell death removes damaged cells and cells no longer critical for development, independently of caspase activation.  Whereas apoptotic cells display condensed nuclei and fragmentation, the telltale signs of necroptosis are plasma membrane permeabilization and mitochondria swelling.  As a regulated cell death mechanism, the process of necroptosis is necessary for maintaining cellular homeostasis during development and as a response to cellular stress conditions.  

Initiation of necroptosis results from blocking pro-survival cues from inhibitors of apoptosis.  Signaling through TNFa (tumor necrosis factor), FASL (FAS ligand) and TRAIL (Tumor...

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...

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