March 12th, 2010
The Novus Product Development Team is excited to announce the launch of a new antibody target not currently available by any other antibody suppliers. The two new antibodies, a human TMEM97 antibody (NBP1-30436) and mouse TMEM97 antibody (NBP1-30437) are of particular interest as TMEM97 is implicated in several cancers, including breast, pancreatic, colorectal and ovarian cancers. In an article published in Cell Metabolism this past July, TMEM97 was also identified as one of 20 genes that are functional regulators of cellular cholesterol homeostasis. Novus’ new human TMEM97 antibody has been successfully tested in both Western blot and immunocytochemistry. The mouse TMEM97 antibody is also useful for Western blot analysis and will be tested in immunocytochemistry within the next week.
In addition to this new product launch, the Novus antibody lab has also been busy with antibody conjugations. A few recently conjugated antibodies include BrdU antibody (NB500-235) to biotin and DyLight 488, Caveolin 1 antibody (NB110-74687) to Biotin, and Jumonji antibody (NB100-2214) to DyLight 649.
Novus’ laboratory technicians have also had their hands full with the QC testing of newly purified antibody lots. A new lot of LC3B antibody (NB600-1384) was tested in human brain lysate and achieved a specific band at 14kDa, consistent with previously purified lots of this antibody. The Tyro-3 antibody (NBP1-28635) was also QC tested on human brain lysate which produced a clear band at 96kDa. One of Novus’ embryonic stem cell markers, an anti-SOX2 antibody (NB110-37235), was also tested in Western blot. The SOX2 antibody was tested on mouse brain lysate where a strong signal at 39kDa was visualized.
The Novus antibody lab has also been busy with new antibody projects. A new autophagy-related antibody is currently in production; more details are expected to be released within the coming month. This new antibody complements Novus’ broad line of over 800 autophagy antibodies.
Tags: Antibodies, Antibody, Antibody catalog, Antibody suppliers
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March 11th, 2010
Recent antibody studies have suggested that nuclear factor κB-inducing kinase (NIK) is inhibited through proteasome-controlled degradation regulated by TRAF/cIAP proteins. cIAP1 and cIAP2 are fairly recent apoptosis inhibitors and represent some of the newer products in our antibody catalog.
NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) is found in practically all mammalian cells and is a transcriptional factor of DNA. It affects the cellular response to a range of stimuli, including free radicals, UV radiation and stress. It plays an important role in the immune response to pathogens, but disruption of the regulatory pathways can lead to cancer and other diseases.
The TNF receptor-associated factor (TRAF) family are adaptor proteins which link various cell receptors to MAPK signaling cascades, thus activating NF-kB. TRAF proteins are important transducers for the TNF and the IL-1/TLR receptor complexes. They play an important role in the adaptive and innate immune responses.
The C-terminals of TRAF2 and TRAF3 interact with receptor domains following ligand-induced oligomerization. They interact with a number of pro and anti-apoptosis proteins, meaning TRAF signaling can promote either cell death or survival. The cell signaling proteins in our antibody catalog are used for both pro and anti-apoptosis studies in both healthy and cancerous cells.
Recent IHC assays showed that NIK degradation was dependent on a TRAF3/NIK, TRAF2/cIAP1 and TRAF2/cIAP2 regulatory complex. cIAP1 and cIAP2 appeared to play redundant roles in NIK degradation, as deactivation of both proteins was required for non-canonical NF-kB activation and B-lymphocyte survival. The NIK pathway is tightly regulated, meaning a single gene is enough to reverse lethal TRAF deficiency.
Our antibody catalog at Novus Biologicals is constantly being updated to reflect new signaling pathway findings.
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March 10th, 2010
MAPK (mitogen-activated protein kinase) antibodies are widely used in cellular research to study these processes, in both healthy and cancerous cells. For example, p38 is a pro-apoptotic factor, and c-Jun NH2-terminal kinase (JNK) regulates cellular longevity and stress resistance. Together they form the JNK/p38 signaling pathway, which is controlled by at least five MAPK cascades.
ASK1, also known as MAPKKK5 and MEKK5, is an apoptosis-regulating kinase which phosphorylates and stabilizes the Daxx protein. The two regulate each other via a positive feedback loop, i.e. higher Daxx levels lead to greater production of ASK1. ASK1 regulates cellular stress, for example that following activation of tumor necrosis factor alpha (TNFα). It is necessary for the sustained activation of JNK.
It has been shown that cancer treatment with TNFα leads to ASK1 activation and accumulation of Daxx. The p53 transcription factor is associated with this pathway, but is easily mutated, and in its mutant form can bind to Daxx. This inhibits the activation of ASK1 and therefore blocks the ASK1 phosphorylation of Daxx. In studies using mutant p53 and Daxx antibody in cancer cells, it was shown that when TM p53 was depleted, the level of Daxx increased and the apoptotic effect of TNFα was restored.
This shows that amplification of the JNK/p38 signaling pathway is important in regulating the cellular response to drug-induced apoptosis. Furthermore, the disruption of this pathway by Tumorigenic mutant p53 can lead to cell stress and tumor development.
The MAPK immunoglobulins that we at Novus Biologicals have in our antibody catalog cover a large number of signaling pathways.
Tags: Antibodies, Antibody, antibody catalogue, p53 antibody
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March 9th, 2010
Apoptosis inhibitors are a well studied group of proteins that have been implicated in the formation of several types of human tumor. The most commonly studied IAP (inhibitor of apoptosis protein) is the Survivin antibody. However, our apoptosis antibody catalog at Novus Biologicals is constantly being updated to reflect the growing list of newer proteins. cIAP1 and cIAP2 antibodies are among the more recent of our anti-apoptosis products.
All IAP proteins are characterized by their ability to suppress cell death via their BIR (baculoviral IAP repeat), a novel domain consisting of around 70 amino acids. Some have an additional CARD and/or RING domains. They prevent apoptosis by inhibition of TNF and other pro-apoptosis proteins. This is done by the binding and inactivation of various caspases (cell death proteases).
The cIAP protein mediates TRAF2 ubiquitination following the receptor binding of TNF-alpha. It has two isoforms, structurally similar to XIAP. Each has three BIR motifs which bind to caspase-3 and caspase-7. cIAP 1/2 form a heteromeric complex which is then recruited to the death receptor TNF-R2 (tumor necrosis factor receptor 2) via the TNF receptor associated factors 1 and 2 (TRAF1 and TRAF2).
cIAP is inhibited by HtrA2 (also called Omi) which acts by catalytic cleaving of the protein. Smac/DIABLO is another negative regulator which works by enhancing the autoubiquitination action of cIAP.
The cIAP1 and 2 immunoglobulins are recent additions to our antibody database at Novus Biologicals, and have been used in studies into human cancers. In January 2010, cIAP2 antibodies were used in a study showing retinoic acid having a possible protective role in the battle against breast cancer.
Tags: Antibodies, antibody catalogue, Antibody suppliers, Survivin antibody
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March 4th, 2010
The antibody lab technicians here at Novus Biologicals are constantly busy conducting purifications, conjugations and QC analysis in order to maintain our antibody catalog of over 100,000 products. Just this past week, the lab team conjugated numerous antibodies and tested several newly purified antibody lots.
A few recent examples of antibody conjugations include conjugating the rabbit polyclonal anti-PINK1 antibody (catalog number NB100-493) and the mouse monoclonal anti-HSD3B1 antibody (clone FDO66Q) (catalog number NB110-78644) to HRP. Horseradish peroxidase (HRP) is the most commonly used enzyme label for antibodies. Its stability, small size and broad specificity make it an ideal label for immunoblotting and immunocytochemistry. The Novus antibody lab also conjugated a large number antibodies to DyLight dyes last week, including rat monoclonal anti-CD105 antibody (clone MJ7/18) (catalog number NB100-77666) and rabbit polyclonal anti-Carbonic Anhydrase IX antibody (catalog number NB100-417).
We at Novus Biologicals adhere to a strict QC analysis process in order to assure our antibodies are validated for the appropriate research applications. Our laboratory technicians conducted numerous Western blots last week. New lots of two GRP78 antibodies (catalog numbers NBP1-06277 and NBP1-06274) were tested on HeLa whole cell lysates where specific bands at 72kDa were visualized for both antibodies. One of our newest antibodies, a LOX Propeptide antibody (catalog number NBP1-30327), was tested in Jurkat lysate and picked up the glycosylated propeptide as well as the proenzyme form. Also tested this week was a SOX2 antibody (catalog number NB110-37235). The new SOX2 antibody lot was tested on mouse brain lysate using a 4-12% MOPS gel. This Western blot analysis produced a clear band at 39kDa.
Check the Novus Biologicals Antibody Blog for frequent updates on our lab and antibody catalog, and to learn more about new antibody developments and recent scientific findings.
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February 26th, 2010
We at Novus Biologicals have a huge range of monoclonal and polyclonal immunoglobulins on our antibody database, and are constantly developing more. Immunoglobulins comprise a number of different classes, and it’s important you select the right one for your needs. Here, we give a brief run-down of antibody classification.
Antibodies are composed of polypeptide units (monomers). Each unit comprises 2 heavy and 2 light chains (linked H-L on either side of the Y) Each chain has a single V (variable) domain, and the V-pairs form the 2 binding sites of the molecule.
The 5 primary classes are IgG, IgM, IgA, IgD and IgG, identified by the type of H-chain polypeptides they have. The H-chains (called g, μ, a, e and d-chains, respectively) allow the immunoglobulins to function in different types of, and particular stages of, immune response. The peptide sequences responsible for this are found mainly in the Fc fragment of the chain. Immunoglobulin light chains comprise only two types – kappa and lambda chains.
Antibody classes also vary in the number of monomers, or Y-units they have. This affects the valency of the protein and varies between species.
The monomeric IgG is the predominant class in humans. Because of its abundance and antigen specificacy it is the preferred class for immunology research; the majority of immunoglobulins in antibody catalogues are IgG. IgA exists in both monomeric and dimeric forms, and is the second most prevalent class, comprising around 15% of the total serum content. It has a primary defence role against local infections and is thought to prevent passage of antigens, rather than destroy them. IgM, a pantamer, can similarly exist in monomeric form.
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February 25th, 2010
Since DNA-damage can lead to the development of tumours, these antibodies are widely used in cancer studies. Cyclin dependent kinases (CDKs), which interact with regulatory cyclins, are essential to the progression of the cell cycle. However, tumours can develop if CDK/cyclin disruption leads to unregulated cell reproduction. Therefore these two proteins are carefully regulated.
p16INK4A is one of several proteins in the INK family which performs this function. It forms part of the pRb (p16-pRb-cyclin D1) pathway. Detailed antibody studies have revealed this pathway is the product of several protein interactions: pRb/E2F, p16INK4A and cyclin D/CDK. CDK inhibits pRb, a tumour suppressor which controls cell cycle progression by E2F inhibition. The E2F transcription factors promote cell replication.
It can be seen that the cell cycle is a complicated network of interactions involving numerous checkpoints. The “traffic flow” leading to the final stage of mitosis is very carefully controlled and can be manipulated to account for changes in cellular and extracellular environments. Any one of these pathways can be implicated in tumour development. Antibody research is heavily involved in working out which pathway/s are involved for particular tumours.
For example, the pRb and p53 (p53-MDM2-p21) pathways are both implicated in tumorigenesis. To establish which proteins were responsible for a particular tumour, assays were conducted using pRb and p53 antibodies specific to the proteins in these pathways. ESCCs (oesophageal squamous cell carcinoma) cells were analysed using IHC assays.
MDM2, p53 and cyclin-D1 were overexpressed, while p21, p16 and pRb were depleted. The overall results showed p16, pRb and MDM2 to be significant risk factors at different stages of cancer development.
We at Novus Biologicals have a wide range of products in our antibody catalogue to aid in cell cycle research.
Tags: Antibodies, Antibody, antibody catalogue, Antibody database
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February 24th, 2010
Not everybody working in immunobiology has an in-depth knowledge of the subject. Some may be students, who are still getting to grips with the discipline at college. Others may have been forced into a swift career change following restructuring at work. For whatever reason, people quite often perform their first antibody assays with only the vaguest knowledge of the underlying concepts. Understanding the molecular structure of a given antibody is fundamental to interpreting its results, therefore we at Novus Biologicals have put a few basic facts together.
Antibodies are glycoproteins composed of one or more Y-shaped polypeptide units. Each of these has two identical heavy (H) and two light (L) chains, forming the left and right binding sites of the Y. The H chains are hinged, and have roughly double the number of amino acids (and therefore molecular weight) of the light chains. The L-chains are non-hinged, and sit inside the ‘arms’ of the Y.
The regions of polypeptide chains are called domains. The amino terminal end of each chain is termed the variable (V) domain. V-domains show considerable diversity compared to the C (constant) domains, and are where antigen binding takes place.
L-chains contain one variable domain VL, and one constant domain CL. The H-chains have one variable domain VH, plus 3 constant domains CH1, 2 and 3. The CH1 and CH2 domains sit either side of the hinge. Each H-L pair forms a single binding site, meaning each antibody unit is a bivalent monomer.
There are five primary antibody classes, and a number of sub-classes. Variations in the heavy-chain polypeptides and number of monomers allow them individual functions in the immune response.
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February 24th, 2010
The Novus antibody lab has been very busy over the past two weeks. Not only has Novus marketed four new antibodies this past week, but the laboratory technicians have purified numerous antibodies, tested new antibody lots in Western blot as a part of Novus’ stringent QC analysis process, as well as custom conjugating various antibodies.
One of Novus’ newly released antibodies, a rabbit polyclonal anti-CLOCK antibody (catalog number NBP1-30326), is perfectly suited for the study of circadian rhythm. The CLOCK protein, also known as KAT13D, belongs to the basic helix-loop-helix (bHLH) family of transcription factors. It is a major protein of interest in the study of circadian rhythm as polymorphisms within the encoded protein have been shown to be associated with abnormal circadian rhythm behavior, such as sleep disorders. This new CLOCK antibody complements Novus’ full line of circadian rhythm antibodies, including BMAL1 antibodies, PER1 and PER2 antibodies, Timeless antibodies and Cryptochrome antibodies.
As a leading supplier of HIF antibodies, Novus is continually monitoring scientific trends for the latest HIF and hypoxia related research reagents. Two out of Novus’ four recently launched antibodies are HIF related. One of particular interest is a new mouse monoclonal anti-Factor Inhibiting HIF-1 antibody (clone 162C) (catalog number NBP1-30333). The Factor Inhibiting HIF-1 protein (Entrez GeneID 55662) facilitates repression of HIF-1 transcriptional activity by binding to von Hippel-Lindau (VHL), which acts as a transcriptional corepressor. VHL inhibits HIF-1 alpha transactivation function by recruiting histone deacetylases. Involvement of VHL in association with FIH provides a unifying mechanism for the modulation of HIF-1 alpha protein stabilization and transcriptional activation in response to changes in cellular oxygen concentration. The second new HIF related antibody is a mouse monoclonal anti-HIF Prolyl Hydroxylase 2 antibody (clone 366G/76/3) (catalog number NBP1-30328). The HIF Prolyl Hydroxylase 2 protein is a HIF-alpha modifier that hydroxylates HIF-1 alpha at Pro(402) and Pro(564), and HIF-2 alpha. It targets HIF through the hydroxylation for proteasomal degradation via the von Hippel-Lindau ubiquitylation complex. Novus also provides the research community with an extensive line of anti-VHL antibodies, which are frequently purchased in tandem with HIF target antibodies.
Lastly, Novus’ fourth new antibody released this week is an anti-LOX propeptide antibody (catalog number NBP1-30327), often referred to as LOPP. This affinity purified rabbit polyclonal antibody recognizes the glycosylated propeptide form of the protein at approximately 35 kDa, as well as the proenzyme form at approximately 50 kDa. Novus’ lab technicians thoroughly tested for the presence of these two forms by running this antibody in Western blot on MDA-MB-231 cell lysates.
Tags: new antibodies
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February 23rd, 2010
IgG is the most prevalent antibody in mammalian tissue, and therefore a major number of the proteins on an antibody database are of this type. The other classes that are studied are IgA, IgM, IgD and IgE. Depending on the tissue and disease being studied, polyclonal and monoclonal versions of all these antibodies are also produced against specific antigens. Cross-reactivity can occur in IHC assays using tissue-derived antibodies; therefore it is common to use fragments, rather than entire Igs as primary immunoglobulins.
Antibody classes are differentiated by their heavy chain structure. The light chains are fairly homogenous, but minor differences can occur among the H-chains of some classes. Therefore these are further divided into sub-classes. IgG has 4 such subclasses. However, they have closely related structures and so cross-reactivity is less common between subclasses than between individual classes. For example, there is little cross-reactivity between IGG1, 2, 3, or 4, but a lot of the phenomenon can occur between IgG and IgA,M,D and E.
IgA is a dimeric molecule that has a mainly protective role. It does not destroy the antigen, but prevents its entry into tissues. As it is often isolated from mammalian secretions, cross-reactivity between immunoglobulins is a problem. In 2001, 3 peptide-based ELISA systems were evaluated for detection of IgG and IgA antibodies specific to the Chlamydia bacterium. The ‘gold standard’ at the time was assay by microimmunofluorescence (MIF). However, it is a difficult process to perform and there was a problem with false positive results owing to cross-reactivity.
The results showed the ELISA assays gave much more accurate results. However, sometimes there’s no choice but to use assays like MIF, and thus antibody suppliers like us at Novus Biologicals are constantly working on specific primary antibodies that offer minimal cross-reactivity.
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