Discovery Antibodies

Bespoke Antibodies

  • Succinocysteine: a PTM and Biomarker of Complex Cellular Dysfunction

    Protein succination is a post-translational modification formed by a reaction between the Krebs cycle intermediate fumarate with protein cysteines to form S-(2-succino)cysteine (2SC). Cysteine predominantly exists in the thiolate form and can act as a reactive nucleophile. Cysteine succination can occur non-enzymatically and is formed by a Michael Addition reaction between fumarate and the free thiol groups of protein cysteines at physiological pH.  The thioether bond of 2SC is considered to be stable to acid hydrolysis and irreversible. Fumarate is a weak electrophile and its modification of thiols is highly pH dependent.  As a consequence, succination can be selective towards functional, low pKa cysteine residues in proteins, such as catalytic cysteine residues in enzymes.

    2-sc

    Figure 1: Diagram showing the Michael Addition reaction to form S-(2-succinocysteine)

    Succination at critical cysteine residues can result in the inactivation of enzymatic activity or protein function in many biological processes. For example, the succination of key components of the iron-sulfur cluster biogenesis family of proteins, Iscu and Nfu1, lead to defects in iron-sulfur biosynthesis required for respiratory chain complexes. Succination of Glutathione has been shown to increase oxidative stress and cellular senescence. The loss of Fumarate Hydratase (FH), the enzyme that catalyzes the reversible hydration/dehydration of fumarate to L-malate, contributes to the accumulation of fumarate and therefore succination. FH deficiency leads to the inactivation of the E3 ubiquitin ligase Keap1 (Kelch-like ECH-associated protein 1) by succination, which promotes the stabilization of Nuclear Factor 2 (NRF2) and activation of the antioxidant pathway. Keap1 also plays a key role in controlling tumorigenesis.

    2SC is considered as a biomarker for mitochondrial stress in obesity, insulin resistance and diabetes. The succination of adiponectin is increased in adipocytes and adipose tissue of type 2 diabetic mice. Adiponectin succination blocks the formation of oligomeric species and secreted forms of adiponectin, which contributes to reduced levels of plasma adiponectin in diabetes. Succination causes irreversible inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) resulting in the loss of activity in muscle of diabetic rats. The elucidation of the succinated proteome will provide an insight into the role of succination in regulatory biology and determine its effect on cellular dysfunction.

    Anti-2SC Antibody (catalogue number: crb2005012) is now available to purchase from our  Discovery® Catalogue

    crb2005017e-anti-2sc-antibody

    Figure 2: Western blot analysis of mouse epithelial kidney (MEK) fumarate hydratase Fh1 fl/fl wildtype and Fh1 -/- knockout cell lysates preparation without and with OVA-2SC antigen. Lane 1: MEK Fh1 fl/fl (100μg) Lane 2: MEK Fh1 -/- (100µg)

     

     

     

     

     

     

    For more information see:

    Frizzell, N. et al (2013). The succinated proteome. Mass Spectrometry Reviews, 33(2), pp.98-109. PMID: PMC4038156 

     Frizzell, N. et al. (2007). Succination of Protein Thiols during Adipocyte Maturation: A BIOMARKER OF MITOCHONDRIAL STRESS. Journal of Biological Chemistry, 282(47), pp.34219-34228. PMID: 17726021

  • CTLA-4 and its Role in Immune Homeostasis

    Cytotoxic T lymphocyte associated gene-4 (CTLA-4), also known as CD152, is a type I glycoprotein belonging to the immunoglobulin superfamily. CTLA-4 is required for immune homeostasis because it functions as a checkpoint for T-cell activation and is a critical inhibitor of autoimmunity. CTLA-4 is constitutively expressed in Foxp3+ regulatory T cells, where it mediates cell extrinsic control of effector responses to regulate immune suppression. In contrast, CTLA-4 expression is only induced in T cells following activation, where CLTA-4 primarily acts as a co-inhibitory molecule to transmit a negative feedback signal. CLTA-4 counteracts with the B7:CD28 pathway by binding to the B7 ligand on antigen-presenting cells with a higher affinity to antagonise CD28 positive co-stimulatory signalling. CTLA4 any also function by down-regulating ligand expression and transmitting inhibitory signals.

    The membrane bound CTLA-4, mCTLA-4, contains an extracellular V domain, a transmembrane domain and a cytoplasmic tail. An alternatively spliced isoform has also been identified to be a secreted soluble form, sCTLA-4, which lacks the transmembrane domain and possess a different cytoplasmic tail. sCTLA-4 can still bind B7 and modulate the strength and durability of the B7:CD28-mediated costimulation. However, sCTLA-4 may also interfere with B7:mCTLA-4 interactions, causing a reduction in the negative signal. sCTLA-4 also exhibits a dual effect on cytokine production to modulate T cell proliferation.  sCTLA4 can inhibit the secretion of activator cytokines IFN-γ , IL-2, IL-7, and IL-13 and activate the secretion of TGF-β and IL-10.

    The levels of sCTLA-4 play a role in determining the fate of immune responses. Low levels of sCTLA-4 have been detected in normal human serum. Increased levels of sCTLA-4 have been observed in several autoimmune diseases such as Graves' disease, Type 1 Diabetes, Coeliac disease, Crohn’s disease, Systemic lupus erythematosus, and Systemic sclerosis. Understanding the role of sCTLA-4 in modulating the immune response may provide an insight to its relevance in autoimmune disease pathogenesis.

    Antibodies against the soluble CTLA-4 isoform are now available to order from our Discovery® catalogue:

    Rabbit Polyclonal anti-sCTLA-4

     4017 anti-human (catalogue number: crb2005177)

    4018 anti-human (catalogue number: crb2005178)

    Mouse Monoclonal anti-sCTLA-4

    CRB4B8 anti-human (catalogue number: crb5005104)

     CRB10D1 anti-human (catalogue number: crb5005105)

    Antibodies were designed and manufactured by Cambridge Research Biochemicals in the United Kingdom. For more information see:

    Wicker et al. (2015). Journal of Immunology. 193:889-900. PMID: 24928993

    Case Study: Analysis of the molecular nature of CTLA-4 using custom Monoclonal and Polyclonal Antibodies

  • c-Fos: A Converging Point for Intracellular Signalling and Transcription Regulation

    The c-Fos proto-oncogene is a converging point for multiple signal transduction pathways from the extracellular milieu into the nucleus. As a member of the Fos family of transcription factors, c-Fos is characterized by a basic leucine zipper for dimerisation and DNA-binding and contains a transactivation domain at the C-terminus.  c-Fos heterodimerizes with c-jun and other related proteins to form Activator Protein-1 (AP-1) complexes that mediate transcription via AP-1 sites. The AP-1 consensus binding site is a palindromic DNA motif TGA(G/C)TCA found in promoters and enhancers.

    c-Fos expression can be triggered by a wide variety of cell-extrinsic and cell-intrinsic signals, including serum, growth factors, tumor promoters, cytokines, and UV radiation. It is considered to be an immediate early gene (IEG) as c-Fos mRNA and protein is rapidly and transiently transcribed upon stimulation, without the need for de novo protein synthesis. c-Fos contains multiple transactivation modules in the N- and C-terminal regions. Its activity is also regulated by kinases, such as MAPK, cdc2, PKA or PKC and its phosphorylation has an influence on its protein stability and transcriptional activity.

    c-Fos is ubiquitously expressed in many cells. As a neuronal IEG, c-Fos has been widely used to facilitate neuronal activity mapping. c-Fos is also involved in various cellular events, including cell proliferation, differentiation, growth, angiogenesis and apoptosis. Dysregulation of the proto-oncogene therefore plays an important factor in carcinogenesis. It can lead to the loss of cell polarity and epithelial-mesenchymal transition to promote tumor invasion and metastasis.

    c-fos-ihc-human-brain-ihc

    Image: Immunohistochemistry analysis of human brain using free floating sections. Immunoreactivity detected in the nucleoplasm in neuronal cells in the cerebral cortex using our anti-c-Fos antibody.

    Antibodies available to purchase via DISCOVERY Antibodies

    Anti-c-Fos antibody  (catalogue number: crb4005003f)

    c-Fos peptide (catalogue number: crb1200272e)

    Alexa Fluor® 488 Anti-c-Fos antibody (catalogue number: crb2115003e)

  • 10% Discount On Your First Purchase from DISCOVERY®

    As an early Christmas present from DISCOVERY® Antibodies & Peptides , we are offering a 10% discount off your first purchase using the code : ENDEAVOUR10

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    The code can be used against all products on the DISCOVERY® Antibodies website and also on our sister site DISCOVERY® Peptides.

     

    Happy Holidays and all the best in the New Year from the Discovery® team!

     

  • Anti-ERK 1/2 Antibody

    Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase (MAPK) super family, playing a role in regulating cell proliferation and apoptosis. ERK1/2 are protein-Ser/Thr kinases that participate in the Ras-Raf-MEK-ERK signal transduction cascade; with 83% shared amino acid identity, ERK1 (44 kDa) and ERK2 (42 kDa) are expressed in almost all tissues and have higher homology in their core regions. Extracellular stimuli such as growth factors, mitogens, cytokines, hormones, and oxidative or heat stress can trigger signal transduction pathways that lead to ERK1/2 activation. For example, human MEK1/2 phosphorylates the effector MAPKs ERK1/2 on Tyr204/187 and Thr202/185 in the TEY sequence to activate its enzymatic function.

    When activated, ERK1/2 preferentially phosphorylate substrates containing the Ser/Thr-Pro motif, with the optimal consensus sequence identified as Pro-Xxx-Ser/Thr-Pro. The ERK1/2 proline-directed kinases phosphorylate a multitude of cytoplasmic and nuclear protein substrates including signaling effectors, protein kinases, receptors and cytoskeletal proteins. In addition to this, ERK1/2 also phosphorylate an array of proteins involved in various processes including cell adhesion, cell cycle progression, proliferation, migration, differentiation, cell survival and metabolism. ERK1/2 can also translocate into the nucleus and phosphorylate many transcription factors. The co-ordinated variations of ERK1/2 signalling from its duration, magnitude, subcellular localization and protein interactions can determine the final outcome of cellular fate. Due to the varied and complex nature of ERK1/2 action, combined with its role in several different signalling transduction pathways, these extracellular signal-regulated protein kinases (ERK1/2) maintain a vital function in cell proliferation and apoptosis.

    Antibodies available to purchase via DISCOVERY Antibodies

    Anti-ERK 1/2 Antibody (catalogue number crb2005025f)

    Alexa Fluor® 488 Anti-ERK1/2 antibody (catalogue number crb1200306e)

    We also stock the ERK1 antigen peptide on our DISCOVERY Antibodies site:

    ERK 1 peptide (catalogue number crb1200306e)

  • Anti-Cyclin D1 Antibody

    Cyclin D1 is a key regulator of cell proliferation as it links the extracellular signaling environment to cell cycle progression.  Cyclin D1 accumulation is the rate-limiting step for cell cycle entry and the transition from G1 to S phase. The levels of cyclin D1 are elevated in G1 phase, where it interacts with the serine-threonine protein kinases, cyclin-dependent kinases (CDK) CDK4 and/or CDK6 to activate its catalytic activity. Active Cyclin D1/CDK complexes then phosphorylates the retinoblastoma protein (Rb).

    Rb inhibits cell cycle progression through its ability to repress E2F transcription factors activity, which is involved in the regulation of genes required for DNA replication and G2/M progression. Phosphorylation of RB (pRb) promotes the release of E2F and subsequently promotes cell cycle progression. Furthermore, cyclin D1 plays a role in maintaining the integrity of the G1/S checkpoint. Cyclin D1 associates with proliferating cell nuclear antigen (PCNA), a component of the DNA replication and repair machinery. During S phase, cyclin D1 down-regulation is necessary for PCNA translocation, DNA repair and initiation of DNA replication.

    Cyclin D1 has been shown to associate with a number of transcription factors in a CDK independent manner to modulate transcription and epigenetic changes. Cyclin D1 contains an LxxLL motif (251-255) that facilitates coactivator recruitment to mediate transcriptional activation. In addition, cyclin D1 contains a repressor domain (142-253) within its central region, which facilitates the interactions with co-repressors to negatively regulate transcription. The levels of cyclin D1 are determined by the rate of expression, protein stability, localization, associations and degradation.

    The diverse roles of cyclin D1 are dependent on its protein level. The various biological processes that cyclin D1 has been implicated in include cell migration, mitochondrial metabolism, cell cycle arrest and apoptosis. Given the pivotal role of cyclin D1 in promoting cell proliferation, aberrant cyclin D1 expression and activity frequently occurs in human cancers. The over-expression of cyclin D1 is predominantly associated with tumorigenesis and metastases.

    Understanding the multifaceted role of cyclin D1 and its dysregulation may provide a better understanding of its involvement in the development and progression of cancer.

    Antibodies available to purchase via DISCOVERY Antibodies

    Anti-Cyclin D1 antibody (catalogue number crb2005024)

    Cyclin D1 peptide (catalogue number crb1200305e)

  • Anti-phosphorylated CDK (pY15) Antibody

    An Introduction to Phosphorylation Site Specific Antibodies

    DISCOVERY® Antibodies is the new catalogue business from Cambridge Research Biochemicals  and within this catalogue several phosphorylation site specific antibodies are featured.

    Protein phosphorylation is a prevalent, versatile and widely studied reversible Post Translational Modification (PTM) and occurs principally on three amino acids: serine, threonine or tyrosine. It is mediated by kinases and phosphatases and plays a critical role in the regulation of many cellular processes including cell cycle, signal transduction pathways and apoptosis.

    Phosphorylation site-specific antibodies which can discriminate between the phosphorylated and non-phosphorylated forms have emerged as reliable tools to enable quantitative and qualitative detection of phosphorylated proteins and can be used for Western blotting, Immunohistochemistry and Immunoprecipitation, ELISA analysis and flow cytometry.

    Anti-phosphorylated CDK (pY15) Antibody

    Cyclin-dependent kinase-2 (CDK2) is a serine/threonine kinase that plays an essential role in coordinating cell cycle. Monomeric CDK2 is inactive, and requires interaction with cyclins for functional activation. The CDK2/Cyclin E complexes are involved in controlling the G1/S transition and initiation of DNA synthesis. Whereas, the CDK2/Cyclin A complexes are involved in the control of progression through S phase and mitosis. The CDK2 is the catalytic subunit of the heterodimeric complexes.

    The CDK2 kinase activity is tightly regulated and depends on the phosphorylation state at 2 major phosphorylation sites, Thr14/Tyr15 (T14/Y15) and Thr160 (T160). CDK2 activation occurs when CDK2 is dephosphorylated at the inhibitory site T14/Y15 and phosphorylated at activating site T160.

    CDK2 contains an N-terminal lobe structure and a C-terminal lobe structure which sandwich the active site. The N-terminal lobe contains a glycine-rich inhibitory element (G-loop) and a unique major helix (C-helix). T14/Y15 residues are located in the flexible G-loop that forms the roof of the ATP-binding site and is important for ATP interaction. Structural studies of pY15/pT160 CDK2/Cyclin/ATP complex show that phosphorylation at Y15 perturbs protein substrate binding affinity due to steric hindrance. This affects correct ATP alignment and binding at the catalytic site.

    Wee1 and Myt1 kinases phosphorylate CDK2 at Y15 and inhibit its activity by preventing ATP binding. The phosphorylation at Y15 of CDK2 is independent of cyclin interaction and precedes the activating CDK2 T160 phosphorylation by CDK-activating kinase complex (CAK), as the CDK2 T160 phosphorylation is dependent on cyclin binding. Phosphorylation at CDK2 T14/Y15 has been shown to stabilize Cyclin E and prevent its degradation.

    The subsequent dephosphorylation of CDK2 at T14/Y15 by the cell division cycle 25 (CDC25) phosphatase promotes CDK2 activation and is the rate-limiting step of the CDK2/Cyclin complex activation process. The CDK2 inhibitory phosphorylation plays an important role during replication stress signaling and in maintaining genome integrity.

    Antibodies to purchase via DISCOVERY Antibodies

    anti-phosphorylated-cdk2-py15-antibody

    Anti-phosphorylated CDK2 (pY15) antibody (50ug) (catalogue number crb2005014e) 

    Anti-CDK2 antibody  (50ug) (catalogue number crb2005068f)

    Phosphorylated CDK2 (pY15) peptide (50ug) (catalogue number crb1200296e)

    CDK2 peptide (50ug) (catalogue number crb1200297e)

    Peptide and Antibody bundles to purchase via DISCOVERY Antibodies

    CDK2 bundle  Includes: Anti-CDK2 antibody and CDK2 peptide

    Phosphorylated CDK2 (pY15) bundle Includes: Anti-phosphorylated CDK2 (pY15) antibody, Phosphorylated CDK2 (pY15) peptide and CDK2 peptide

    DISCOVERY Antibodies also offer a wider selection of Post Translational Modification specific antibodies. Please visit the website for citrullinated and other phosphorylated antibodies.

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