Antibody News

ABC Membrane transporters and the role of MRP1 in drug resistance

Thursday, May 26, 2016 - 10:39

ATP-binding cassette (ABC) transporters, alongside ion channels and aquaporins, are ubiquitous membrane-bound proteins that move substrates across extra and intra cellular membranes.  Multidrug resistance-associated protein 1 (MRP1) is a member of the superfamily of ATP-binding cassette (ABC) transporters, and in humans is encoded by the ABCC1 gene. In addition to endogenous substrate exchange, ABC transporters also participate in the movement of drugs and chemicals across cell surface and cellular organelle membranes, suggesting that defects in these genes are of great importance in cancer therapy and pharmacokinetics.  In fact, MRP1 was initially identified due to its ability to yield multidrug resistance in lung cancer cells.  MRP1 is one of many genes that may alter a tumor cell to expel cancer drugs and deem them ineffective. Specifically, MRP1 acts as a pump, disposing of cytotoxic chemicals from tumor cells. Drug resistance in chemotherapy is a constant...

Why LC3B Antibodies Make Ideal Autophagosomes Membrane Markers

Tuesday, May 24, 2016 - 15:07

The human form of microtubule-associated protein light chain 3 (LC3) is expressed as 3 splice variants; LC3A, LC3B and LC3C (He et al., 2003). LC3B is a subunit of the MAP1A and MAP1B microtubule-binding proteins and plays a central role autophagosome membrane structure. This ubiquitin-like modifier is known to be involved in early stages of autophagosome formation and specifically with phagophore membrane elongation. LC3B also interacts with autophagy receptors such as p62/SQSTM1 and NBR1 during substrate selection for autophagic degradation. (Johansen et al., 2011).

Researchers using LC3B knock out mice to determine if LC3 is required for autophagy found that the mice do develop normally, likely due to a compensatory autophagy mechanism by other members of the MAP1LC3...

Controls for HIF-1 Alpha's WB, ICC-IF, IHC, IP & FLOW Analysis

Friday, May 20, 2016 - 13:57

Tips on positive and negative controls for HIF-1 alpha antibodies is one of the most Frequently Asked Questions on Hypoxia and HIFs. Here are top 5 suggestions from Novus Biologicals:

  1. The degradation of HIF1 alpha is the most common issue which often results in either a weaker specific signal and/or the appearance of multiple degraded protein bands in Western blot (WB). Degradation may be avoided by preparing the lysates quickly (on ice/at 4˚C) after the collection of cells or tissues, preferably in a hypoxic chamber. For differentiating the target band from degradation (40-80kDa) or dimer (200+ kDa) bands, we suggest including a control based on true hypoxia or hypoxia mimetic samples (e.g. cells treated with CoCl2). Novus offers the following ready to use lysates for HIF-1 analysis.

NFkB and p62 Both Activate and Regulate Inflammation

Thursday, May 19, 2016 - 14:53

Nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) is a protein complex that regulates DNA transcription and is a critical regulator of cell survival. NFkB has long been known as a primer of inflammation, however researchers are now finding that NFkB may also regulate over-inflammation via a novel mitophagy pathway (Minton, 2016).

NFkB proteins are structurally homologous with retroviral oncoproteins originally known as v-Rel, and are now classified together as NF-κB/Rel family proteins. These include NFkB1 p50/p105, NFkB2 p52/p100, RelA/NFkB p65, RelB and c-Rel, which can function together as dimeric transcription factors to regulate gene expression via the NFkB signaling pathway (Gilmore 2008).

...

The TGR5 Receptor and metabolic disease

Monday, May 16, 2016 - 15:16

The bile acid-responsive G-protein-coupled receptor TGR5 is widely distributed across the human body - including the endocrine glands, adipocyte cells, muscles, immune organs, spinal cord, and the enteric nervous system. G protein coupled receptors (GPCRs) are incredibly versatile signaling molecules that are activated by a number of ligands, which in turn regulate various signaling pathways. Select G proteins stimulate activity, whereas others have inhibitory effects.  The effect of TGR5 activation depends on the tissue where it is expressed and the signaling cascade that it induces.  Specifically, bile acids initiate the activation of the TGR5 receptor.  BA’s primary role are to facilitate the uptake and absorption of lipids, making their receptors important for bile acid synthesis and metabolism, as well as integration of multiple homeostatic functions in the liver and gastrointestinal tract.  Since its discovery, TGR5 has been...

Wnt-5a Antibodies Help Understand Wnt Mediated Signaling in Embryogenesis and Various Diseases

Friday, May 13, 2016 - 13:57

Wingless-Type 5A (Wnt-5a) is a member of the WNT family of secreted signaling proteins that regulate many important developmental processes including cell proliferation, migration, differentiation, fate determination and embryonic patterning. WNT signal proteins affect the cell via three known WNT signal transduction pathways. The canonical WNT signaling pathway regulates gene transcription, the non-canonical planar cell polarity pathway regulates cytoskeletal formation, and the non-canonical Wnt/calcium pathway regulates cellular calcium levels.

Wnt5a signals through both the canonical and non-canonical WNT pathways and plays an essential role in regulating developmental pathways during embryogenesis. Mutations in the Wnt5a gene are associated with Robinow syndrome (Roifman et al., 2015) and may also play a role in oncogenesis.

Recently, Wnt5a has been shown to regulate embryonic cell proliferation and differentiation in...

Understanding the relationship between HIF-1 alpha, Hypoxia and Epithelial-Mesenchymal Transition

Tuesday, May 10, 2016 - 14:08

Epithelial-mesenchymal transition (EMT) is a natural process by which epithelial cells lose their polarity and intercellular adhesion, and gain the migratory invasive properties of mesenchymal stem cells that can differentiate into a variety of cell types. EMT is critical to many developmental processes including embryo development and wound healing. However, EMT is also a fundamental step in the initiation of metastasis during cancer progression.

Hypoxia is known to induce EMT via HIF-1 alpha induction. Researchers have used HIF-1 alpha antibodies (NB100-105), (NB100-449) and (NB100-479) to help identify the molecular mechanisms...

How DOPA Decarboxylase Affects Neurotransmitter Synthesis

Friday, May 6, 2016 - 13:11

DOPA decarboxylase (DDC) is responsible for catalyzing the conversion of aromatic amino acids into their corresponding amines during the synthesis of several important neurotransmitters. Specifically, DDC catalyzes the decarboxylation of L-DOPA to dopamine, L5-HTP to serotonin, L-histidine to histamine, phenylalanine to phenethylamine, L-tyrosine to tyramine, and tryptophan to tryptamine.

A number of mutations in the DDC gene can cause aromatic L-amino-acid decarboxylase deficiency (AADCD) (Gücüyener et al., 2014). AADCD is an inborn error in neurotransmitter metabolism that leads to combined serotonin and catecholamine deficiency. DDC's critical role in neurotransmitter synthesis makes it an important target in the study of Parkinson's disease, depression and other neuroscience related diseases.

Parkinson's patients experience motor system symptoms due to reduced levels of dopamine in the substantia nigra portion of the brain. The conversion of L-DOPA...

STING in Innate Immunity and Cancer: What’s the Buzz About?

Tuesday, May 3, 2016 - 15:08
STING (STimulator of INterferon Genes protein) acts as a sensor of cytosolic DNA. Bacteria/Virus or self-derived DNA in the cytosol activates the STING pathway and promotes the production of type I interferons (IFN-alpha and IFN-beta). STING also participates in cell death signaling through its association with MHC-II and the ERK pathway. STING has been suggested to interact with DDX58/RIG-I, MAVS, SSR2, RNF5, TRIM56, TBK1, IFIT1 and IFIT2. It generally localizes to the cytoplasm and membranes of the cell, ER, and mitochondria; however, in response to DNA stimulation, it translocates to the perinuclear region and interacts with TBK1 kinase. STING’s phosphorylation by TBK1 at Ser-358 results in STING activation. STING executes its role by sensing and binding cyclic di-GMP/c-di-GMP and cyclic GMP-AMP/cGAMP. This binding results in the activation of NF-kappa B and IRF3 transcriptional...

EZH1 has more to offer than gene repression

Friday, April 29, 2016 - 14:45

EZH1 is part of the Polycomb-group family of proteins, which are responsible for remodeling chromatin in genes and modulating epigenetic silencing during development.  Specifically, EZHI is a component of PRC2, or polycomb repressive complex-2.  PRC2 interacts and modifies the histone “H3”, and is critical in maintaining gene repression.  EZH1 has the ability to mono-, di- and tri-methylate 'Lys-27' of histone H3 to form H3K27me1, H3K27me2 and H3K27me3, respectively.  EZH1 is also implicated in the maintenance of embryonic stem cells, including their differentiation and renewal.    

Margueron et al took a closer look at the mammalian homologs of EZH1 and EZH2 in order to learn more about their similarities and differences during development (1). They found that while both Ez proteins form PRC2 complexes, they carry out different repressive roles.  Ultimately, both protein complexes are efficient at...

Choline Acetyltransferase (ChAT) – a useful Immunohistochemical marker for morphological studies of neurons

Tuesday, April 26, 2016 - 07:45

Choline Acetyltransferase (ChAT) is the enzyme that is responsible for biosynthesis of the neurotransmitter acetylcholine. The majority of acetylcholine is synthesized locally at nerve terminals where ChAT catalyzes the transfer of an acetyl group from acetyl coenzyme A to choline, a process that takes place in a single step. ChAT is expressed by cholinergic neurons in the central nervous system (CNS) and peripheral nervous systems (PNS) (4), which are distributed in many different regions of the brain, spinal chord, and retina and participate in learning, memory, movement, and vision (2,4).

ChAT antibody staining is frequently used in morphological studies of cholinergic cell populations. In the mammalian retina, for example, ChAT is expressed by subsets of both amacrine and ganglion cells (2). These ChAT-positive neuronal populations can be identified using...

The role of PARP-1 in the repair of single stranded break (SSB)

Friday, April 22, 2016 - 13:50

PARPs (poly ADP ribose polymerases) are DNA repair enzymes that promote single stranded break (SSB) repair by binding to DNA at the sites of SSBs and recruiting repair machinery. In humans, the PARP superfamily consists of 17 members, of which five play known roles in SSB repair. PARP-1, the most well-studied family member, is required for base excision repair and is thought to be responsible for 90% of PARP activity (5).

PARP inhibitors are a class of pharmacological agents that have been used to treat certain types of cancer, and are designed to induce cell death by preventing cancer cells from repairing damaged DNA. PARP inhibitors have been tested in various combination therapy contexts. For instance, one recent study compared radiation-induced cytotoxicity in cell culture and xenograft models of pancreatic carcinoma and found that PARP inhibition with radiation increased cell death in vitro and reduced tumor...

Using RPE65 as a tool to investigate ocular gene therapies

Tuesday, April 19, 2016 - 13:17

While not life threatening, blindness and retinal disease are profoundly debilitating and greatly affect quality of life.  Understandably, gene therapy has been subject to controversy given it’s potential effects on the rest of our cellular processes.  However, a genetically diseased eye being an isolated organ quickly becomes a promising prospect for such therapies.  Specifically, RPE antibodies are powerful diagnostic tools to test the viability of these clinical treatments. 

The RPE65 protein is organized into a thin layer of cells known as the retinal pigment epithelium (RPE). This RPE provides support to the retina, which sits behind the eye and has sensitivity to light. A mutation in RPE65 leads to retinal degeneration and LCA (Leber congenital amaurosis - an autosomal recessive childhood blindness), which results in a lack of 11-cis retinal productions and an inability to efficiently form the visual pigments rhodopsin and cone opsin....

HIF-2 alpha: HIF1A's Homologue with Similar and Divergent Functions

Thursday, April 14, 2016 - 13:19

HIF-2 alpha is a member of the heterodimeric hypoxia-inducible factors/HIFs family (HIF-1, HIF-2, and HIF-3) which contains a common beta subunit but differ in their alpha subunits. Also called as EPAS1 or Mop2, HIF-2 alpha regulates cellular adaptation to hypoxia which is involved in several biological processes such as angiogenesis, cellular survival/proliferation, energy metabolism, erythropoiesis, extra-cellular matrix functions, invasion/ metastasis, iron metabolism, pH regulation, multidrug resistance, stem cell properties. The expression of HIF-2 alpha is regulated through hypoxia-dependent protein stabilization with the help of proteins

IRE1 alpha dependent apoptotic-signaling pathway

Monday, April 11, 2016 - 12:47

Despite in depth characterization of the role of IRE1 alpha (inositol-requiring enzyme 1 alpha) in activating the unfolded protein response (UPR) in the ER - little is known about the molecular mechanisms by which this ER protein has shown to regulate intracellular calcium levels and subsequent apoptosis. Intracellular calcium homeostasis is fundamental to many physiological processes, and an increase in Ca2+ is associated with both the early and late stages of apoptosis. A Nature article fleshed out a pro-apoptotic IRE1alphaTRAF2JNK pathway that has potential to be activated by prolonged ER stress. Specifically, prolonged IRE1-mediated activation may promote apoptosis by degrading the mRNAs that encode essential cell-survival proteins. Additional research studies have proposed similar pathways that bring in other pro apoptotic...

Caspase-3- A marker of programmed cell death

Thursday, April 7, 2016 - 13:44

Caspases, or cysteine-dependent aspartate specific proteases, are a family of enzymes crucial for initiating and executing apoptosis within a cell, an important biological event especially during organ development (1). Environmental cues and cellular signals trigger the initiation of the programmed cell death cascade primarily through proteolytic activation of the caspases (1). One specific effector caspase is caspase-3, a protein that is cleaved and thus activated upon the initiation of apoptosis. Cleaved caspase-3 propagates an apoptotic signal through enzymatic activity on downstream targets, including poly ADP ribose polymerase (PARP) and other substrates (2). In cell biology, caspase-3 antibodies that detect both uncleaved and cleaved versions of the enzyme are strong indicators of cell death induction. For example, caspase-3 antibodies are widely...

The subunit RelA(p65) mediates NF-kB signal transduction in multiple ways

Thursday, March 31, 2016 - 11:00

RelA (also known as p65) is an NF-kB family member and a subunit of the NF-kB transcription factor complex.  The mammalian NF-kB family has five members (NF-kB1, NF-kB2, RelA (p65), RelB, and c-Rel), each of which contains an N-terminal Rel homology domain. Active NF-kB protein complexes are dimeric (hetero- or homo-), and are made up of two family members. NF-kB signaling is activated in response to many different types of stimuli and modulates transcription of numerous downstream targets. NF-kB-mediated signaling plays known roles in inflammatory and immune responses (Hayden and Ghosh, 2008), as well as neuronal development and synaptogenesis (Boersma et al., 2011; Gutierrez et al., 2005).  Aberrant NF-kB activity has also been linked to various human diseases including a number of cancers (Perkins, 2012).

In the central nervous system, NF-kB activity is dependent on translocation to the nucleus, where the complex binds to DNA...

Three things everyone studying autophagy should know

Friday, March 25, 2016 - 09:36

Novus Biologicals' antibodies are the gold standard to monitor autophagy and detect LC3 expression. The recently published Guidelines for the Use and Interpretation of Assays for Monitoring Autophagy (3rd Edition) comprehensively details methods to monitor autophagy in cell or tissue samples. Importantly, these guidelines also provide key considerations for data interpretation and tips to creating better western blot data.  

Use fresh samples and avoid multiple freeze thaws to improve detection of LC3-I when possible.

Why? LC3-I is less stable and more susceptible to freeze thaws and degradation in SDS compared to LC3-II. Moreover, most LC3 antibodies are less sensitive to LC3-I than...

Multifaceted Roles of Matrix Metalloproteinase-2 (MMP2) in Normal and Disease State

Monday, March 21, 2016 - 13:25

MMP2 is a 72 kDa enzymatic protein and it belongs to matrix metalloproteinases (MMPs), a heterogenous family of zinc/calcium-dependent TIMPs (tissue inhibitors of matrix metalloproteinases) regulated matrix-degrading endopeptidases which are classified into collagenases (MMP-1, -8, -13, -18), gelatinases (MMP-2, -9), stromelysins (MMP-3, -7, -10, -11), elastase (MMP-12), and membrane-type matrix metalloproteinases (MT-MMP-1 through -5) (1). MMP2 involves in extracellular matrix metabolism and cleaves type IV collagen along with degrading the already denatured collagens. TIMP2 and MT-MMP1 (membrane type MMP, MMP14) regulate the activity of MMP2, and the cleavage of pro-MMP2 to active-MMP2 is dependent upon MMP14. MMP2 is expressed ubiquitously and it implicates in a diverse array of functions such as vasculature remodeling, angiogenesis, tissue repair, tumor invasion, inflammation, and atherosclerotic plaque rupture. Mutations in MMP2 gene has been linked to...

Glial Fibrillary Acidic Protein (GFAP), The Most Popular Astrocyte Marker

Wednesday, March 16, 2016 - 13:43

GFAP, a class-III intermediate filament, is a 50kDa protein which is found in the mature and developing astrocytes in the CNS, non-myelinating Schwann cells in the PNS, enteric glial cells (enteric nervous system/ENS), ependymal cells, and radial glia of the developing brain. GFAP antibodies are the most popular marker for astrocytes in neurological studies and along with its breakdown products (BDPs), GFAP has been proposed as a useful candidates for biofluid-based markers for numerous neurological conditions especially during traumatic brain/spinal cord injury and stroke [1]. Moreover, neuro-oncology studies have established a positive reaction to GFAP in astrocytomas, ependymoma, and astrocytic cells of mixed gliomas, subependymal giant cell astrocytoma, pleomorphic xanthoastrocytoma, astroblastoma, and gliosarcoma [2].

...

Caspase 3 - a Reliable Marker for Index of Apoptosis Induction

Friday, March 11, 2016 - 15:16

Caspase-3 is one of the most important players in apoptosis signaling. It is synthesized as an inactive 32 kDa pro-enzyme and upon direct activation by Caspase-8, -9 or -10, it gets processed into its active forms, the p17-20 and p10-12 subunits. The latter are responsible for the cleavage of PARP (poly ADP-ribose polymerase), actin and SREBP, which are associated with apoptosis [1]. Because of its role in coordinating the destruction of cellular structures, Caspase 3 is often regarded as an executioner or effector caspase and considered as a marker in various immunoassays for apoptosis related experiments.

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Caspase 3 antibody

 

Tips on choosing an ideal loading control antibody for Western Blotting

Monday, March 7, 2016 - 12:56

Western blotting is one of the most commonly used antibody assay techniques in cell and molecular biology research since its development over three decades ago, and is considered the gold standard for protein detection and quantification.

When performing a Western Blot, it is crucial to ensure equal loading of protein samples and protein transfer through the use of a loading control antibody. beta-Actin, GAPDH and alpha Tubulin are well known housekeeping proteins that are commonly used as loading controls. While there are various options available, researchers often face some challenges in choosing a proper loading control antibody as expression of housekeeping proteins can be influenced by sample type, treatment or other...

FSH R - a hormone receptor critical for both female and male reproductive systems

Monday, February 29, 2016 - 15:33

FSH R, or follicle-stimulating hormone receptor, is a transmembrane G-protein coupled receptor that is expressed in the ovaries, uterus, and testes. The ligand for this receptor, considered to be the central hormone of mammalian reproduction, is called follicle stimulating hormone (FSH) [1]. In females, FSH R is essential for proper ovarian development and follicle maturation. In males, it is required for normal spermatogenesis. Linkage analysis revealed that in females, a mutation in the extracellular ligand binding domain of FSH R segregated with a disease called hypergonadotrophic ovarian dysgenesis [2]. In males, with a mutation in the intracytoplasmatic loop that leads to ligand-independent constitutive FSH R activation, spermatogenesis is sustained in the absence of gonadotropins [3]. Based on these examples, it is...

Include tissue controls in every IHC experiment to instill confidence in your results

Wednesday, February 17, 2016 - 12:15

Interpretation of immunohistochemistry (IHC) data is difficult in the absence of appropriate controls.

To confirm staining specificity and instill confidence in your results, a positive and negative tissue control should be routinely included in IHC experiments.

Positive Tissue Control Negative Tissue Control

Control slides

 

Need help identifying a positive and negative control for your IHC experiment...

Tyrosine hydroxylase - a marker for dopaminergic neurons in the central nervous system

Monday, February 15, 2016 - 15:48

Tyrosine hydroxylase is a member of the aromatic amino acid hydroxylase (AAAH) family.  It is expressed throughout the central nervous system (CNS) and catalyzes the conversion of tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), which can be, through a series of downstream enzymatic reactions, processed into the neurotransmitter and signaling molecule dopamine. Dopamine can then be further altered to produce norepinephrine or epinephrine. Tyrosine hydroxylase is the rate limiting enzyme in this pathway, also referred to as the catecholamine synthesis pathway. 

Antibodies that detect tyrosine hydroxylase are often used to identify dopaminergic neurons in the CNS.  In the mammalian retina, for instance, a subset of dopaminergic amacrine cells that form a single synaptic strata in the inner retina specifically express tyrosine hydroxylase and are often identified through tyrosine hydroxylase antibody staining (Wulle and...

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