2012 ARCHIVE

December 2012: We release a new mouse monoclonal to High Mobility Group Box 1 (HMGB1) protein, called MCA-1F3. HMGB1 is an important and abundant molecule found in the nucles of eukaytic cells where it associates with histones and DNA, but it also has a role as one of the “Damage Associated Molecular Pattern Molecules”. These so-called DAMPs are proteins released from damaged cells which bind to Toll like receptor (TLR) family members. HMBG1 is released from necrotic but not apoptotic cells and binds to TLR4 and so induces an inflammatory response. We also release a mouse monoclonal antibody to Aldolase C, a major cytoplasmic enzyme found concentrated in astrocytic cells. This is MCA-4A9, and it was raised against an N-terminal peptide of human Aldolase C.

September 2012: We release new antibodies to visinin-like protein 1 (VSNL1). This is a small calcium binding protein related to calmodulin which is expressed in very high levels in many neurons. These are two mouse monoclonals, MCA-2D11 and MCA-3A9 and a rabbit polyclonal is in progress. VSNL1 antibody can be used to identify and classify neurons in sections and in tissue culture, and has been shown to be released from damaged and degenerating neurons following traumatic brain injury and in Alzheimer’s disease. In other news, our CEO, Gerry Shaw, is now co-founder and scientific advisor of a new company, Iron Horse Diagnostics, which will use EnCor antibodies and biomarker assays in the clinical diagnosis of Amyotrophic Lateral Sclerosis (ALS, a.k.a. Lou Gehrig’s disease) and other CNS damage and disease states. Various studies have used EnCor assays to show that the neurofilament subunit pNF-H is released into blood and CSF at greatly elevated levels in ALS patients, and that the levels detected are of prognostic and diagnostic utility. The other co-founder, president and CEO of Iron Horse is Dr. Robert Bowser of the Barrow Neurological Institute in Phoenix, Arizona, and the major facility of the new company will be in the Phoenix area.

August 2012: We have now generated an excellent monoclonal antibody to Ubiquilin 2, a protein implicated in familial forms of Amyotrophic Lateral Sclerosis (Lou Gehrig’s disease). This is clone MCA-6H9. We also release a monoclonal to the RRM domain protein Fox1, a.k.a. ataxin 2 binding protein 1 or A2BP1, clone MCA-1G10. Fox1 is a close relative of Fox2 and Fox3/NeuN, all three being homologues of the Fox1 originally identified in C. elegans. Fox3/NeuN antibody is widely used marker of neuronal nuclei. Like NeuN/Fox3, antibody to Fox1 is an excellent marker of neuronal nuclei in the central nervous system. Note we also market antibodies to NeuN/Fox3, specifically our mouse monoclonal antibody MCA-1B7 and rabbit polyclonal antibody RPCA-Fox3.

July 2012: We have now generated two new monoclonal antibodies to the lysosomal associated membrane glycoprotein LAMP1. These are MCA-5H6 and MCA-6E2, and both work well on blots, revealing a diffuse band in the range of molecular weights 90-120kDa, as expected, since LAMP1 is heavily and variably glycosylated. Both antibodies also work well in immunohistochemistry, revealing early endosomes and lysosomes cleanly and strongly. These antibodies can be used to identify and track lysosomes, as western blotting standards and to monitor the level of lysosomal activity.

June 2012: We have now manufactured antibody preparations from the hybridoma lines we licensed from the laboratory of W. Clay Smith and collaborators, of the Department of Ophthalmology at the University of Florida. These are two excellent antibodies to rhodopsin, MCA-A531 and MCA-B630 and an excellent antibody to enolase 1 (a.k.a. enolase α and non-neuronal enolase). This is MCA-253, which binds to the N-terminal peptide of enolase 1 and does not cross-react with the very closely related molecules enolase 2 (a.k.a. neuron specific enolase and enolase γ and enolase 3 (a.k.a. enolase β). We also release an excellent antibody to visual rod arrestin (a.k.a. arrestin-1 and S-antigen), clone MCA-S128, also from this group.

May 2012: We plan to release yet more antibodies this month; Firstly we release a chicken polyclonal antibody to mCherry, CPCA-mCherry. Also we now supply some of the excellent antibodies developed by Dr. David Muir in the University of Florida Department of Pediatrics, including his widely used peptide antibodies to matrix metalloprotienase II (MMP2- RPCA-MMP2) and growth associated protein 43 (GAP43, RPCA-GAP43).

April 2012: We release several new antibodies this month- Firstly there is MCA-4C4, an excellent mouse monoclonal to Lamin A/C which works well both on immunoblots and in immunocytochemistry. This antibody can be used to visualize the nuclear lamina and as a western blotting control, since Lamin A/C levels are quite stable in cells and tissues under most conditions. Secondly we release MCA-6H11, a mouse monoclonal antibody to heat shock protein 27 (Hsp27), an ubiquitous and important chaperone protein. Thirdly we release RPCA-mCherry, our affinity purified polyclonal antibody to mCherry, an engineered form of a coral red fluorescent protein. Finally we release another mouse monoclonal antibody to MAP2, MCA-4H5, an excellent marker of neuronal perikarya and dendrites. This antibody complements our already released MAP2 mouse monoclonal MCA-5H11 and our extremely popular chicken polyclonal to MAP2, CPCA-MAP2.

March 2012: We continue to test products from other companies with our antibodies as a means of getting further information on both, of potential benefit to both us and our collaborators. Recently we obtained some specialized tissue culture dishes from Akron Biotechnology LLC, a company located in Boca Raton, Florida. This company makes various kinds of 3-D tissue culture scaffolds. We obtained two Akron PolyFiber 24 well plates, one which had aligned nanofibrils and the other in which the fibrils were randomly organized. The fibrils are about the same size as extracellular matrix proteins which they are intended to mimic. We were able to grow many kinds of cell line on these plates with no problems, and were able to see that the the cells became strongly orientated along the aligned fibrils, but were randomly oriented in plates with randomly oriented Polyfibers. Some images are here, and show cells double labeled with our popular chicken vimentin antibody CPCA-Vim and our new mouse monoclonal antibody to actin, MCA-5J11. We also found out that we will be working on a newly funded research project with P2D Bioscience, an established drug discovery and biomarker company located in Cincinnati, OH. We will contribute antibodies and expertise to this project. Finally we license a series of mouse monoclonals to rhodopsin, arrestin/S-antigen and Enolase 1 from the laboratory of W. Clay Smith, of the Department of Ophthalmology at the University of Florida. We have fully characterized these excellent reagents and will be ready to market them soon.

February 2012: We release a new mouse monoclonal to actin, one of the most abundant and highly conserved proteins of vertebrates. Our antibody,

MCA-5J11, binds the protein products of all six mammalian actin genes strongly and cleanly on western blots. Unlike some companies we could name, we actually have data to prove this (see here). Since the antibody binds these six abundant proteins which are found essentially all kinds of mammalian cell and tissue extract, we believe it will be an excellent protein standard for quantification of bands on western blots, comparable to our very popular monoclonal antibody to glyceraldehyde 3-phosphate dehydrogenase, MCA-1D4. The actin antibody also works in immunofluorescense, and stains the submembraneous cytoskeleton, filopodia and stress fibers in HeLa, 3T3 and similar cells. If you would like a free sample of either of these antibodies email mike@encorbio.com including your shipping address. We also initiate OEM supply agreements with two further reagent companies, namely United States Biological and Biomolecular Integrations. United States Biological is a large and well established vendor located in Swampscott, Massachusetts, while Biomolecular Integrations is a relatively new company located in Little Rock, Arkansas.

January 2012: Our founder and CEO Gerry Shaw obviously had time on his hands over the holidays and was wondering how to make best use of our new Nanodrop spectrophotometer. As most readers will know, spectrophotometers measure the absorbance of protein and nucleic acid solutions and are widely used to quantify the levels of these substances in solution. The Nanodrop machines are particularly quick and convenient for this purpose. Protein absorbance determinations are usually done at a wavelength 280nm, at which only the amino acids Tryptophan, Tyrosine and Cystine (i.e. two disulfide linked Cysteine residues) absorb. Unfortunately all three are rather rare amino acids, and it is quite possible for a protein to have very low or unusually high levels of each or any of these, so that the same amount of different proteins can have wildly different absorbance values. To use a spectrophotometer with accuracy on a pure protein it is therefore necessary to know the absorbance based on the content of these three amino acids. Gerry wrote a useful on line calculator which can integrate the absorbance of a protein, determined from the sequence, with the OD value measured, to get an accurate determination of the amount of protein. The page is here, and other useful protocols, many of which are now linked to by other companies and sources, are available here.