Antibody News

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.

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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? 

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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. 

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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.

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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. 

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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.

The role of c-Fos in the regulation of the JC virus gene transcription

Thursday, April 20, 2017 - 11:10

c-Fos is a member of the AP-1 transcription factor family under the Fos protein family umbrella, alongside Fra-1, Fra-2 and Fos-B.  Also in the AP-1 transcription family are the Jun proteins, c-Jun, Jun-B and Jun-D.  Each member of the AP-1 transcription family is a phosphonuclear protein composed of a carboxy-terminal leucine zipper domain, a basic domain and an amino terminal transactivation domain. Together the Fos and Jun families compose a dimeric complex that binds to response elements on DNA in order to regulate gene expression, cell proliferation, cell differentiation, tumorigenesis and more.  Their induction can be catalyzed by a number of signals, including cytokines and growth factors, stress and viral infection. ...

The advantages and applications of using tissue microarrays

Monday, April 17, 2017 - 15:04

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. 

tissue comparsion slide

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An overview of permeabilization in immunocytochemistry (ICC)

Thursday, April 13, 2017 - 09:12

Immunocytochemistry (ICC) can be a very effective method for visualizing the localization and behavior of intracellular proteins, however the protocol for each ICC experiment should be optimized specifically to the cell being examined.  Permeabilization, or the puncturing of the cell membrane, is an extremely important step in detecting intracellular antigens with a primary antibody because it allows entry through the cell membrane. Permeabilization is introduced after cells have been prepared with a fixative agent to initiate protein cross-linking, such as formaldehyde or ethanol. However, determining the amount of exposure to a permeabilization agent is crucial, seeing as a hard tissue organ may require a longer incubation over a soft tissue organ and treating your sample incorrectly will lead to unreliable results. 

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Pathway Highlight: Which caspase substrates contribute to the morphological features associated with apoptosis?

Tuesday, April 11, 2017 - 09:20

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...

Key Targets in Apoptosis, Necroptosis, and Autophagy

Sunday, April 2, 2017 - 14:16

Cell death/recycling pathways such as apoptosis, necroptosis, and autophagy are an integral part of the growth, development, homeostasis as well as the pathophysiology in the life of living organisms. These signaling pathways are highly regulated and some of their key regulatory targets are discussed below.

Apoptosis

Apoptosis, programmed cell death, is primarily characterized by the activation of caspases which further regulate the mass cleavage of proteins and DNA. Some of major the proteins responsible for various apoptotic events are:


Initiator Caspases
(-2, -8, -9, -10)

In apoptosis, initiator caspases are involved in the upstream events of death receptor (extrinsic)- or mitochondrion-dependent (intrinsic) signaling pathways. They...

Why you should use a Rabbit Monoclonal Antibody

Thursday, March 30, 2017 - 11:50

Primary antibodies are becoming increasingly popular investigate tool to use in research, given the breadth and selection of product on the market. Not only are antibodies raised in a variety of hosts, they also have proven reactivity with a wide range of species applying to many research fields and models. From here one can select a desired clonality, choosing polyclonal or monoclonal depending on the target of interest.  With monoclonal antibodies, it can be taken one step further to hone in on a specific clone of interest. With all of the different options available, it can be difficult to choose your ideal antibody.  Choosing the correct make up of an antibody will no doubt have an effect on your experimental outcomes,...

Potential therapies for human ovarian cancer surrounding the PI3K/AKT/mTOR pathway

Tuesday, March 28, 2017 - 09:47

Mammalian target of Rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth, proliferation, motility and survival. Acting downstream of AKT, also a serine kinase, mTOR is composed of the mTORC1-Raptor complex and the mTORC2-Rictor complex.  mTOR has been implicated in many cancers, including ovarian cancer, due to the effect of mTOR inhibitors on tumor progression.  In addition, mTORC2 has a positive feedback effect on AKT behavior, which may explain its rapamycin resistance.  Rapamycin is a common drug used as a targeted therapy for mTOR inhibition, however it is not successful in all cancer types. In Ovarian Cancer specifically, the PI3K/AKT/mTOR pathway is the most frequently altered pathway, with PI3K, AKT and mTOR expression levels correlating with survival and tumor growth.  In the following articles, an mTOR antibody is used to investigate the efficacy of two...

What are the major differences between Apoptosis, Necroptosis & Autophagy?

Friday, March 24, 2017 - 13:41

Apoptosis is a form of programmed cell death which is mediated by cysteine proteases called caspases. It is an essential phenomenon in the maintenance of homeostasis and growth of tissues, and it also plays a critical role in immune response. The cytomorphological alterations and the key features of apoptosis are listed below:

apoptosis

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Process

Active, physiological or pathophysiological

Induction stimuli

Oxidative stress, death receptor ligands, chemotherapy

Ultrasensitive IHC Detection with HRP-Polymer Conjugates

Thursday, March 23, 2017 - 14:54

Signal amplification methods are widely used in immunohistochemistry (IHC) for detection of rare epitopes and low abundance antigens. While many of these techniques such as the avidin-biotin complex (ABC) method improve staining, they frequently require additional steps and result in higher background staining.  Blocking endogenous biotin, a requirement of using ABC reagents, may not sufficiently remove residual activity in frozen tissue sections and tissues high in biotin including the liver and kidney.

FABP1/L-FABP antibody

Figure 1. FABP1/L-FABP was detected in paraffin-embedded sections of human kidney using Mouse Anti-Human FABP1/L-FABP Monoclonal Antibody (Catalog # MAB2964) at 1 μg/mL for 1 hour at room temperature followed by incubation for 30 minutes at room temperature with Anti-Mouse/Rabbit IgG VisUCyte HRP Polymer Antibody (Catalog...

The use of apoptosis antibodies and controls in cell death research

Monday, March 20, 2017 - 15:06

Apoptosis is a method of programmed cell death that is notably characterized by a morphological change in cellular nuclei and membrane appearance.  Not to be confused with necrosis, apoptosis is a pathway that is induced by a variety of factors that activate cysteine proteases known as caspases to lead the cell to its ultimate death versus natural death of a cell. While excessive apoptosis can be detrimental, it can also be part of our immune response or in protection of cells that have been damaged by disease or toxic stimuli.  Using antibodies against apoptotic pathway proteins is an effective way to investigate the role of apoptosis in a number of experimental models.  However, it is important to use antibodies against well-defined key apoptotic players and to integrate controls when possible. 

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