Carbonic Anhydrase IX (CA-IX) is an enzyme that is induced under hypoxic conditions. This enzyme is rarely present in normal cells and is responsible for controlling tumor pH. CA-IX is a transmembrane glycoprotein of the zinc metalloenzyme family. This family displays 15 isoforms in human tissues. Carbonic Anhydrase IX functions to convert carbonic acid present in hypoxic cells into a biocarbonate and a proton. Due to the hypoxic conditions found in many tumors CA-IX has become a marker of hypoxia and expression has been associated with several tumors such as lung, brain, colorectal, ovarian, renal, breast, and cervical to name a few. Carbonic Anhydrase IX can be regulated through Hypoxia Inducible Factors. HIF proteins cause upregulation of the CA-IX gene to stabilize the microenvironment of tumor/hypoxic cells. The upregulation of the CA-IX gene is caused by Hypoxia Inducible Factor (HIF) undergoing specific hydroxylations dependent on oxygen conditions within the cell. Once the hydroxylation occurs HIF-1 alpha can interact with the beta domain on the Von Hippel-Lindau protein. In the presence of oxygen, HIF proteins will undergo rapid degradation. In the absence of oxygen, HIF can activate a signal cascade to transcribe several targets such as Carbonic Anhydrase IX. Since many tumors induce hypoxic conditions, CA-IX is being used as a marker. Studies are being performed to look at levels of hypoxia within certain tumors, as hypoxic tumors often are more aggressive and have a lower survival rate. In the publication “The key hypoxia regulated gene CAIX is upregulated in basal-like breast tumours and is associated with resistance to chemotherapy” by Tan, E. Y., Yan, M., et al. (2009) in the British Journal of Cancer, 100(2), 405-411. the authors suggest that CA-IX positive tumors may be due to an advanced hypoxic response and that continued research on the HIF pathway and targets regulated by this pathway such as CA-IX are pivotal in future cancer research.
Archive for the ‘Uncategorized’ Category
Carbonic Anhydrase IX and Hypoxic Response: A Path to Tumors
Wednesday, November 30th, 2011Analyzing LC3 in Western blot
Monday, November 28th, 2011Microtubule-associated protein light chain 3 (LC3) is considered one of the definitive markers of autophagy, and its use is widespread in labs throughout the world. Despite its popularity, there are several considerations when employing LC3 antibodies in immunoassays, and in particular Western blots.
LC3 is expressed as a propeptide, and is subsequently cleaved to form LC3-I. Initiation of autophagy causes the conversion of LC3-I to LC3-II via the addition of a phosphatidylethanolamine (PE) group to the C terminus. The PE group increases the rate of band migration in an SDS-PAGE gel, likely due to its hydrophobic nature; this modification commonly manifests as the appearance of a doublet in a Western blot. The lipophilic character of the PE group also facilitates the insertion of LC3-II into the membranes of autophagosomes, and as a result LC3-II is degraded as autophagosomes are turned over. It is common practice to consider an increase in LC3-II band intensity and a decrease in LC3-I expression as the hallmark of autophagy, but increases in LC3-II can be caused by enhanced autophagosome synthesis or reduced autophagosome recycling, and can make interpreting LC3 staining very misleading.
Analysis of LC3 Western blots is further complicated by a difference in the affinities of antibodies for LC3-I and LC3-II, and by the marked difference in LC3 expression in varying cell types and tissues. Due to these factors, a consensus has emerged that the total amount of LC3-II only should be evaluated and compared to a loading control as a means of assessing autophagy. In addition, many investigators include control extracts collected from cells treated with various inhibitors in order to determine the efficiency of autophagic flux in their experimental samples. The LC3 expression pattern in these controls ideally indicates the cause of LC3-II accumulation (or its absence) depending on the type of inhibitor used. One last consideration is the measurement of autophagic flux over a period of time as opposed to harvesting extracts from one static point. For example, although the shift from LC3-I to LC3-II is evident following short periods of stress, the LC3 signal disappears after extended starvation in many cell types.
Please feel free to contact our technical support staff or more information on LC3, autophagy, and for assistance designing the best controls for your experiment.
The ABCA1 Antibody and ABC Transporter Research
Thursday, August 25th, 2011The ABC (ATP-binding cassette) transporters are a superfamily of transmembrane proteins which play an essential role in membrane transport in both prokaryotes and eukaryotes. In humans, ABC transporters have been implicated in many conditions. For example, ABCA1 antibody research has shown mutations in the ABCA1 gene can cause Tangier disease, while the ABCB family causes multiple drug resistance. We at Novus Biologicals have a large antibody database of ABC transporters and related proteins, for a variety of species and applications.
ABC transporters use energy harnessed from ATP hydrolysis, to carry out a range of functions, including the translocation of lipids, drugs and metabolic substrates across intracellular and extracellular membranes. However, research has also shown that some proteins do not have a membrane transport function but play other roles within the cell, such as DNA repair and RNA translation.
In prokaryotes, ABC transporter proteins may be either importers (mediating uptake of nutrients) or exporters (removing drugs and toxins). In eukaryotes, the majority are exporter proteins. However, not all the ABC transporters on our antibody database play a direct role in transport. CFTR (Cystic Fibrosis Transmembrane Regulator), for example, uses ATP hydrolysis to regulate opening and closing of ion channels.
ABC transporters are classified according to their ATP-binding domains. There are 48 known ABC transporters in humans, divided into seven subfamilies. ABCA1 antibody research has revealed this protein plays a major role in homeostasis, regulating efflux of phospholipids and cholesterol to apolipoproteins such as apo-A1, reversing formation of foam cells and promoting HDL formation. The ABCA family contains some of the largest ABC transporters – in excess of 2100 amino acids.
2Q Novus Travel Grant Winner Announced
Thursday, July 7th, 2011Novus is happy to announce the winner of our 2nd Quarter Travel Grant, Dr. Evelyn Schlenker of University of South Dakota’s Sanford School of Medicine. Dr. Schlenker will now receive a $500 travel grant towards airfare and a conference registration of her choice.
Dr. Schlenker became eligible for this quarterly drawing by simply submitting an online product review. The next quarter’s winner will be announced in early October, so be sure to get your review submitted by the end of September! Please email rewards@novusbio.com if you have any additional questions.
Win $500 to attend your next Conference on Novus!
Monday, June 6th, 2011Novus is launching a new Travel Grant offer for researchers who provide feedback on our antibodies.
For each complete product review that you submit, you will be entered into a drawing for a $500 travel grant! We will select a winner every 3 months – giving you 4 chances for you to win each year! The winner will receive a $200 airline voucher and $300 towards registration for a scientific conference of your choice.
Complete reviews will require a descriptive summary of the experiment and results, regardless of whether they are positive or negative, and a high quality image. However, researchers may also submit reviews for previously untested species and applications that are being tested under the Innovators Reward Program. Therefore, you could earn Innovators Rewards, Novus points toward a future discount, and be entered to win the Travel Voucher, all by submitting one review.
Entry is simple, so login and submit a review today!
Featured Product Citation using Novus’ hTERT Antibody
Monday, May 2nd, 2011In the article “Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis” from the December 2009 edition of the Journal of Immunology, Dr. Qunzhou Zhang et al. cite using Novus’ mouse monoclonal Telomerase Reverse Transcriptase Antibody (cat# NB100-317). Specifically, Novus’ hTERT antibody is used for immunofluorescent staining on human gingiva-derived mesenchymal stem cells (GMSC). Novus’ hTERT specific antibody helps the authors demonstrate that the GMSC’s are in fact a new population of precursor cells “which exhibit several unique stem cell-like properties as MSCs derived from bone marrow and other postnatal tissues.”
Novus Biologicals offers a complete line of Telomerase Reverse Transcriptase antibodies for Western blot, immunostaining, flow cytometry and immunoprecipitation. Novus also offers hTERT lysate, hTERT antibody pack, hTERT RNAi, and ancillary reagents for your hTERT research. Finally, you may find a wide range of other cellular markers and antibodies related to DNA repair online at www.novusbio.com.
Zhang Q, Shi S, Liu Y, et al. Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis. J Immunol. 2009 Dec 15; 183 (12): 7787-98. [PMID: 19923445]
Research Using the 160kDa Medium Neurofilament Antibody
Friday, April 29th, 2011Neurofilaments (NFs) are intermediate filaments found almost exclusively in neuronal cells, and play an essential role supporting the cytoskeleton. In vertebrates they are composed of three intertwining polypeptide subunits of varying length and molecular weight – the light, medium and heavy NF chains. We at Novus Biologicals have an extensive range of neurofilament antibody products on our antibody database.
The three NF chains share similar structures and base sequences, but vary in length and sequence at both the N and C-termini. In NF-H and NF-M subunits, the C-termini form extensions which link neurofilaments both to each other and to other cytoplasmic proteins.
Neurofilament antibody research advanced a long way with the advent of SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), which enables proteins to be separated and accurately measured according to their electrophoretic mobility. NF-L, M and H subunits are 68-70kDa, 145-160 kDa and 200-220kDa in size respectively, with protein sizes varying according to species.
Neurofilament antibody products are widely used in neuropathology immunostaining assays, as overexpression of neurofilaments is a feature of many diseases. They are routinely used in NF cell marker studies.
In 2002, Duplan et al used 160kDa neurofilament antibody assays to study antitumor FGF (fibroblast growth factor) activity relative to FGF receptor expression in medulloblastoma tumor variants. More recently the 160kDa neurofilament antibody has been used in a study linking human TDP-43 expression in mouse motor neurons to growth deformities and early death. The NF products on our antibody database are suitable for use in a wide range of assays including western blot, immunocytochemistry, immunohistochemistry and immunoprecipitation.
The c-Myc Antibody – A Major Tool in Cancer Research
Friday, April 22nd, 2011C-Myc is a widely expressed transcription factor, regulating cellular differentiation, proliferation, cell cycle progression and pro-apoptotic gene expression. The c-Myc antibody is widely used in cancer research, as a number of human tumors have been attributed to altered c-Myc expression.
In normal tissue c-Myc expression is tightly regulated, only occurring when cells actively divide. However, in cancer cells genetic aberrations cause the gene to be expressed in an uncontrolled fashion. C-Myc antibody studies have revealed abnormal expression of the protein in 90% of gynecological cancers and 80% of breast tumors. Overexpression has also been reported in 70% of colon cancers and 50% of hepatic carcinomas, as well as a number of hematological cancers. Around 100,000 cancer deaths a year can be attributed to the c-Myc gene in the US alone.
C-Myc is a basic helix-loop-helix-leucine zipper (b-HLH-LZ) protein. It can be activated by forming a heterodimer with Max, another b-HLH-LZ monomeric protein. It is thought transcription occurs through a combination of histone acetylation and direct interaction with the transcription apparatus. Max has been known to bind to certain Mad proteins, forming heterodimers which block c-Myc transcriptional activity in association with other proteins, including histone deacetylases and Sin3.
C-Myc antibody research has revealed cellular proliferation and cell cycle progression may be controlled by phosphorylation at Thr58/Ser62, via glycogen synthase kinase 3, cyclin dependent kinase, ERK2 and JNK (C-Jun N terminal Kinase) interaction. Among the c-Myc antibody products in our antibody catalog, we at Novus Biologicals have a phospho c-Myc antibody specific to Thr58/Ser62 phosphorylated c-Myc. It does not react with the protein in its non-phosphorylated form.
How PARP Antibody Assays Aid Apoptosis Research
Friday, April 15th, 2011The PARP (Poly(ADP-ribose) polymerase) protein is a zinc-dependant nuclear enzyme whose main role is to detect and repair DNA single-strand breaks (SSB). However, PARP antibody research has revealed there are at least 17 PARP proteins, which also play a major role in apoptosis (programmed cell death) and post-translational modification. We at Novus Biologicals have an extensive range of PARP antibody products in our antibody catalogue.
PARP is activated in response to metabolic, radiation or chemically-induced DNA SSBs. Once a break is detected, the enzyme binds to the DNA and begins synthesis of a poly ADP-ribose chain, or PAR, utilising NAD as a substrate. This is done in partnership with other repair proteins including DNA ligase III, DNA polymerase beta and the scaffolding protein XRCC1, resulting in a base excision repair. Once this is completed, PAR degradation takes place.
Recent PARP antibody studies have revealed PARP enzymes play an essential role in apoptosis, in a number of ways. Catalytic activity has been shown to be stimulated by the oligomerization of PARP-2 with PARP-1, which also plays a role in transcription by chromatin remodelling. Caspase 3 proteolytic cleavage of PARP is a key event in apoptosis.
Also, following DNA cleavage initiated by apoptotic enzymes, PARP can deplete cellular ATP to aid DNA repair; however, this also causes lysis and cell death. During DNA repair, overexpression of PARP can lead to cellular NAD-depletion, leading in turn to progressive ATP depletion, inhibition of glucose oxidation and necrotic cell death. PARP can also directly initiate apoptosis, and initiate expression of inflammatory proteins. Recently, the PARP antibody reagents in our antibody catalog have proven important to breast cancer research.
Epitope Tags and the V5 Tag Antibody
Wednesday, January 12th, 2011We at Novus Biologicals have a large antibody catalogue targeted to epitope tags. They include C-Myc, GFP, HA, DYKDDDDK, and V5 Tag antibody reagents. The V5 tag antibody recognises the artificially engineered V5 epitope, which is formed of amino acid residues 95 to 108 of the simian virus 5 RNA polymerase alpha subunit.
Epitope tagging is widely used in proteomics and genetic studies. The epitope is, of course, the portion of the antigen protein to which the antibody binds. An epitope tag is an artificial epitope that is recognised by a common antibody. The genetic sequence of the tag is engineered using recombinant DNA techniques, and grafted into the protein of interest by placing the encoded sequence of the epitope into a matching open reading frame. The antibody raised to that epitope then recognises it, enabling the tagged protein to be isolated, purified, visualised and analysed by immunochemical methods.
Epitope tags are short sequences of around 10 to 15 amino acids. Our V5 tag antibody is 14 amino acids long and is normally used in this form, though a shorter sequence of nine amino acids has been used. Like all anti-epitope tag sequences it is engineered onto the N- or C- terminus of the protein of interest. This minimises structural disruptions which may affect the protein’s function, and allows immunochemical visualisation and analysis.
In 2009 V5 tag antibody assays were part of a study demonstrating that distinct classes of chromatin-interacting membrane proteins have a shared function at the end of mitosis. Another study linked oestrogen receptor pathways to the reading disorder dyslexia. We have 21 V5 tag antibody products in our antibody catalogue, giving plenty of scope for researchers.