Antibody News

Wet or dry protein transfer: Which one to choose for your western blotting?

Tuesday, February 12, 2019 - 10:50
Novus Biologicals western blot resources page

By Jamshed Arslan, Pharm. D., PhD.

Western blotting involves three steps: 1) separating proteins by size through gel electrophoresis 2) transferring separated proteins to a solid support (typically, a PVDF membrane) and 3) using antibody to mark your protein(s) of interest in the mixture of proteins. The second step of moving negatively charged proteins from the gel to a blotting membrane (PVDF) can be performed in dry or wet conditions.

Dry/semi-dry transfers

Dry conditions are famous for easiness and speed. Reagents, power packs and wiring involved in dry transfers are less messy and therefore, potentially less hazardous. They allow blotting of several...

Animal Models to Study Autophagy

Tuesday, February 5, 2019 - 09:03
Autophagy Signaling Pathway, Novus Biologicals

By Christina Towers, PhD

What is autophagy?

Autophagy is the catabolic process that degrades cytoplasmic material via the lysosome. In the beginning... the process of macroautophagy was originally characterized in yeast where the core autophagy proteins or Apgs were identified. In this first system, the multistep process of phagophore initiation and elongation as well as the conjugation machinery that mediate efficient autophagosome completion were determined and cloned to assess function. The mechanisms of starvation induced autophagy as well as many of the selective autophagy processes were also hashed out in...

Sample collection from mammalian culture cells for kinomic analysis

Tuesday, January 29, 2019 - 10:10
ERK1/2/5 Signaling Interactive Pathway

By Jamshed Arslan Pharm.D., PhD.

Introduction

Kinome describes kinases, and kinomics refers to the kinase signaling. Studying the effects of reagent (exogenously applied growth factor or inhibitor) on kinase activity is a common experiment in cell, developmental and integrative biology laboratories. This blog offers a cost-effective and robust method of obtaining adequate amount of sample for kinomic analysis from mammalian culture cells like HEK cells after treatment with reagent. Using this method, you can easily get over 50 μg protein sample in 50 μl volume from a confluent well of a standard 6-well plate with 0.5 x 106...

Best Methods to Induce and Inhibit Autophagy Pharmacologically

Tuesday, January 22, 2019 - 09:33
VPS35 expression in endosomes and lysosomes of human U-2 OS cells, ICC

By Christina Towers, PhD

Autophagy facilitates the degradation and recycling of damaged cytoplasmic material. The multistep process consists of a double membrane structure called an autophagosome that engulfs proteins and organelles and then fuses with lysosomes to facilitate their breakdown. Autophagy is tightly regulated by nutrient availability and is negatively regulated by the robust nutrient sensor mammalian target of rapamycin (mTOR). Starvation, either by glucose deprivation or amino acid depletion, will cause a marked increase in autophagy, best quantified by an increase in autophagic flux. Likewise,...

Phagocytes in Multiple Sclerosis: Myelin uptake leads to oxysterol-induced activation of liver X receptors, LXRs

Tuesday, January 15, 2019 - 08:41
Myelin basic protein (MBP) expression in cerebellum tissue IHC

By Jamshed Arslan Pharm.D., PhD.

Myelin is a cholesterol-rich layer around nerves. Damage to this protective layer and the consequent slowing down of nerve impulses are hallmarks of multiple sclerosis (MS) and other demyelinating diseases. Myelin breakdown alters plasma levels of 27-hydroxycholesterol (27OHC) and other CNS-specific oxysterols, which are natural ligands of transcription factors called liver X receptors (LXRs). Since myelin phagocytosis is a source of intracellular oxysterols in the resident microglia and infiltrating macrophages, a team of scientists in Europe decided to test the hypothesis that myelin uptake and subsequent...

mTOR Signaling and the Tumor Microenvironment

Tuesday, January 8, 2019 - 09:10
mTOR signaling pathway poster

By Yoskaly Lazo-Fernandez, PhD

The mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that, as a member of two distinct intracellular protein complexes, mTORC1 and mTORC2, regulates protein synthesis, autophagy metabolism, proliferation and survival. More and more research is showing how the catalytic activity of both mTOR complexes plays an important role in cancer biology. Not only mTOR is upregulated and contributes to the development of numerous cancer types, but it also contributes to the angiogenic and immune responses within the tumor microenvironment (TME)1.

...

NOX2 oxidase is a potential target for broad-spectrum antiviral therapies

Wednesday, January 2, 2019 - 09:24
TLRs signaling pathway – interactive

By Beth Melson, MS

Viral infection induces the production of reactive oxygen species (ROS) by inflammatory cells, the majority of which is produced by the NOX2 oxidase. While NOX2 oxidase contributes to the clearance of bacteria and fungi, its role in viral infection is less clear, and in fact seems to contribute to increased viral pathogenicity. Recently, it was demonstrated that viruses, including influenza A, induce NOX2 oxidase production through suppression of TLR7 sensing single-stranded RNA (ssRNA)1. Therefore, inhibition of the NOX2 oxidase pathway represents an opportunity for viral...

TMEM 119 is a specific marker of microglia cells

Thursday, December 20, 2018 - 08:58
P2Y12/P2RY12 expression, human cerebral cortex shows cytoplasmic positivity in microglia, IHC

By Jennifer Sokolowski, MD, PhD.

Microglia are a major immune-cell component in the brain. They ingest and degrade dead cells, debris, and foreign material and interact with other immune cells to orchestrate central nervous system immune responses.1,2 Microglia appear to play a critical role in modulating normal physiologic immune functions as well as the immune response in disease states. In order to define the role of microglia, we need specific markers that allow distinction of microglia from other cell types; fortunately, researchers have validated a new microglia-specific marker,...

The role of Wnts in neuroinflammation

Tuesday, December 18, 2018 - 08:35
TRPM2 expression in rat hippocampus neurons, IHC

By Michalina Hanzel, PhD

The multifaceted roles of the Wnt family of glycoproteins have been extensively characterized throughout embryonic development and adult homeostasis. The highly conserved, cell- and tissue- specific proteins orchestrate processes ranging from neural induction, cell proliferation and migration to adult neurogenesis and neuronal maintenance and regeneration. Recently, Wnt proteins have been proposed to have key roles in regulating pathological processes in the brain, especially those observed in neurodegenerative disorders, often resulting from pathological states of neuroinflammation.

...

RNA-binding protein Staufen1 conspires with Atxn2 in stress granules to cause neurodegeneration by dysregulating RNA metabolism

Tuesday, December 11, 2018 - 11:19
PCP4 expression in mouse cerebellum Purkinje neurons, IHC

By Jamshed Arslan Pharm.D.

Spinocerebellar ataxia type 2 (SCA2) is a movement disorder characterized by neurodegeneration. The cause of this autosomal dominant disease is a mutation in the RNA processing gene Atxn2, which leads to polyglutamine (polyQ) expansion in ATXN2 protein. The association between mutant polyQ proteins, neurodegeneration, and dysfunctional autophagy has been extensively studied in amyotrophic lateral sclerosis (ALS), autism, and Huntington’s disease, but not in SCA2. It is noteworthy that ATXN2 is widely expressed in our nervous system, partly as a constituent of stress granules (SGs), which...

Flow Cytometry Basics for the Non-Expert

Tuesday, December 4, 2018 - 10:35
Flow cytometry dot plots, histograms, density plots

By Jody Bonnevier, PhD.

Flow cytometry: A Challenging Interdisciplinary Application

Flow cytometry has long been the cell analysis method of choice for immunologists, however more and more disciplines have recognized its powerful and flexible capabilities, and want to harness the advantages of this multiparameter single-cell analytical tool. Nowadays, the benefits of flow cytometry for cell phenotyping extend from basic to clinical research and diagnostic applications as well as to drug discovery and development.

In basic research, the same flow cytometry techniques used to phenotype T cell subsets can be applied to defining protein...

Losing memory: Toxicity from mutant APP and amyloid beta explain the hippocampal neuronal damage in Alzheimer's disease

Tuesday, November 27, 2018 - 13:42
Map2 and Fox3/NeuN expression in rat hippocampus, IHC

By Jamshed Arslan Pharm.D.

Alzheimer's disease (AD) is an irreversible brain disorder that destroys memory and thinking skills. The telltale signs of AD brains are extracellular deposits of amyloid beta (a polypeptide that comes from proteolysis of precursor protein APP) and intracellular neurofibrillary tangles. The consequent neurodegeneration affects regions associated with memory and cognition, the most noteworthy of which is the hippocampus. It goes without saying that generating immortalized primary hippocampal neurons that could express AD-related protein(s) would immensely...

Application Focus: I see an increase in LC3, now what?

Monday, November 19, 2018 - 13:05
LC3A expression in HeLa cells, chloroquine treated, ICC

By Christina Towers, PhD.

Autophagy is highly conserved and tightly regulated process that all cell types use to recycle nutrients, particularly in the instance of stress1. As a result, even small changes in signaling pathways, gene expression, or drug treatments can alter autophagy. If you stumble into the field of autophagy by happenstance and hypothesize a change in autophagy with your favorite cell manipulation of choice, you probably read a handful of publications and came to the conclusion that you should assay LC3 levels to test this hypothesis. In fact, LC3 expression can be a good readout for autophagy, but researchers should be...

E-syt in Autophagosome biogenesis: What is the source of it all?

Tuesday, November 13, 2018 - 13:44
Calreticulin expression in vesicles and endoplasmic reticulum of HeLa cells, ICC

By Christina Towers, PhD.

Macroautophagy is a cellular recycling process that requires the formation of double membrane structures to engulf and degrade damaged cytoplasmic material. The pathway involves over 20 core autophagy proteins (

Negative feedback regulation of EPAS1 gene in non-small cell lung cancer through DNA methyltransferases

Tuesday, November 6, 2018 - 09:29
HIF-1 alpha expression in human lung tissue, IHC

By Jamshed Arslan Pharm.D.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. NSCLC cells show increased expression of HIF-1alpha and HIF-2 alpha, which help cells overcome hypoxia. However, NSCLC samples have shown decreased mRNA levels of HIF-2 alpha-encoding gene called EPAS1. The mechanism of downregulation of EPAS1 transcription in NSCLC is unknown. A team of researchers from various institutes in Shanghai and Yangzhou, China, suspected transcriptional inactivation through DNA...

Nickel induces migratory and invasive phenotype in human epithelial cells by epigenetically activating ZEB1

Tuesday, October 30, 2018 - 09:05
Epigenetic mechanisms Novus Biologicals

By Jamshed Arslan Pharm.D.

Nickel (Ni) is a naturally abundant metallic element. It is a major component of stainless steel, coins, and many other items of daily use. Disturbingly, Ni exposure is associated with cancer and several diseases of the lung, kidney and cardiovascular system. Despite being carcinogenic, Ni’s ability to cause DNA mutations and induce oxidative stress is low. To explain this paradox and investigate how the impact of carcinogen exposure may last for months or even years, researchers at the New York University and University of Virginia examined Ni-induced changes in transcription and cellular regulation in human epithelial cells from lung and urinary...

A link between Autophagy and Apoptosis: Chat with First Author Brent E. Fitzwalter

Tuesday, October 23, 2018 - 09:00
Apoptosis poster Novus Biologicals

By Christina Towers, PhD.

Autophagy is a cellular recycling process and most often a pro-survival mechanism that regulates cellular homeostasis. On the contrary, apoptosis is an extensively conserved and elaborate programmed cell death process, and it is well established that the two processes are often opposing forces within the cell. Despite the agreed upon notion that autophagy can protect cells from apoptosis, the mechanistic link has yet to be elucidated. This is especially important given the 60 (and growing) number of clinical trials that are currently underway inhibiting autophagy in cancer in combination with other chemotherapies and targeted therapeutics.

The field is hopeful...

The inflammasome: an inflammation-initiating machine, Novus Biologicals

Wednesday, October 10, 2018 - 09:34
ASC/TMS1 expression in Hela cells ICC

By Stephanie Melchor

The inflammasome is a large, multimeric protein complex found primarily in innate immune cells, which are white blood cells that can attack a wide range of pathogenic threats. Three main elements make up the inflammasome: 1) the sensor – a cytosolic pattern recognition receptor that senses pathogen- or damage-associated molecular patterns (PAMPs and DAMPs), 2) an adaptor protein called ASC, and 3) a caspase effector that cleaves and activates pro-IL-1 beta and pro-IL-18.

Prior to inflammasome activation, a cell must first be "primed" with...

Epigenetic Control of Autophagy

Tuesday, October 9, 2018 - 08:48
G9a expression in human MCF7 cell line ICC

By Christina Towers, PhD.

In the last 20 years, epigenetic regulation has become front and center for almost all fields of biology and its role in diseases like cancer and neurodegeneration are being heavily studied. Epigenetics can be defined as a change in phenotype without a change in genotype that is caused by remodeling the chromatin landscape and is often mediated by changes in histone marks, like methylation, acetylation, phosphorylation, SUMOylation, ubiquitination, and glycosylation to name a few. The cellular recycling process of autophagy is among one of the many processes...

Bad news for stomach cancer: BAMBI protein inhibits gastric carcinoma via TGF-beta/epithelial-mesenchymal transition signaling

Tuesday, October 2, 2018 - 09:03
Collagen-I expression in rat colon tissue IHC

By Jamshed Arslan Pharm.D.

Gastric carcinoma is the second leading cause of cancer-related deaths worldwide. One of the key features of gastric carcinoma is acidosis, which promotes growth and metastasis of gastric cancer cells by inducing HIF-1alpha, TGF-beta, and epithelial-mesenchymal transition (EMT) signaling pathways. Gastric cancer cells have a reduced expression of bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a pseudoreceptor that negatively regulates TGF-beta signaling by inhibiting the...

Chemotherapy-induced metastasis: An unexpected foe?

Tuesday, September 25, 2018 - 09:02
Mena plasma membrane, cytosol and focal adhesion expression in human cell line U-2 OS ICC

By Yoskaly Lazo-Fernandez, PhD

Introduction

Evidence has accumulated recently indicating that common cancer therapies might stimulate metastasis in a significant number of cancer patients1. In fact, neoadjuvant chemotherapy (NAC) drugs, which are administered preoperatively as a first line of treatment, have been associated with increased infiltration of endothelial progenitor cells and macrophages in primary tumors, resulting in higher angiogenesis and tumor regrowth2. Mechanisms by which these cellular injury responses induce metastasis, both in preclinical in vivo models, and in human...

Advancing CAR T Therapies with CRISPR/Cas9

Tuesday, September 18, 2018 - 09:27
Expression of CD4 and CD8 in peripheral blood mononuclear cells ICC

By Victoria Osinski

Scientists have turned to gene editing techniques to modify patients’ T cells to combat cancer, but are often limited by factors including cost, low cell yields, or availability of expertise for therapy development. Interest lies in developing “off-the-shelf”, universal, donor cells, which require thorough T cell modifications to make this approach safe and feasible. Currently, scientists are leveraging the use of the gene editing tool clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to advance CAR T therapies.

"Off-the-shelf" donor T cells requires a combinatorial approach

Patients...

How a cell "reaches" out for help

Tuesday, September 11, 2018 - 09:30
Expression of alpha-synuclein in rat neuron-glial cultures ICCchloroquine-treated, ICC

By Christina Towers, PhD.

Parkinson's disease is a debilitating neurodegenerative condition defined by the accumulation of alpha-synuclein-containing (alpha-SYN) intra-cytoplasmic inclusions, called Lewy bodies. The protein degradative processes of autophagy, and most specifically chaperone mediated autophagy and mitophagy, play an important protective role against this disease by targeting neurotoxins for lysosomal mediated degradation1. Alpha-SYN neurotoxins most commonly affect neurons, but they can also...

Combined NOTCH/MAPK inhibition: A promising new anticancer approach

Tuesday, September 4, 2018 - 11:23
MAPK signaling pathway Novus Biologicals

By Yoskaly Lazo-Fernandez, PhD

Introduction

Cancer invasion requires cancerous cells to overpower the barriers of the extracellular matrix and reach adjacent tissues. Invading cells undergo a process called epithelial-mesenchymal transition (EMT), which enables them to dedifferentiate into a highly invasive mesenchymal stem cell-like phenotype. Interestingly, once the cells have reached their new location through metastasis, they undergo a reverse process named mesenchymal-epithelial transition (MET) which allows the solid tumor to reorganize in the distant organ.

Molecular pathways involved in cancer invasion have thus become important targets for anticancer...

Rescuing motor neuronal loss in amyotrophic lateral sclerosis by inhibiting phosphatidylinositol-4 kinase

Tuesday, August 28, 2018 - 08:58
Novus C. elegans antibodies

By Jamshed Arslan Pharm.D.

Motor neurons control our ability to speak, walk, swallow, and even, breath. People with a motor neuron disease called amyotrophic lateral sclerosis (ALS) cannot perform these primal functions optimally. A point mutation (P56S) in VAMP associated protein isoform B (VAPB), a protein associated with the membrane around the endoplasmic reticulum, causes late-onset familial ALS8 by prompting VAPB to aggregate and lose its job of preventing buildup of abnormal proteins. To study ALS8, C. elegans is one of the animals of choice due to its simple neurophysiology: a single worm has only 302 neurons and 113 of them are...

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