Ramblings

Hi, this is the first post in an occasional series which will appear on the EnCor website. It comes out of requests by various individuals who liked to get my writings on various topics, which I was reliably informed were sometimes informative, sometimes amusing and sometimes even accurate. This all grew out of journal clubs I used to run in the University of Florida, which were focused on cutting edge and significant research. In order to prepare people for these I used to send out an email with a pdf of the paper and a short synopsis of the work, why it was important and what were the possible implications. I could not help but make these emails extremely irreverent and I am told amusing. In fact many individuals wanted to get the emails but had no intention of ever going to the journal club. So hopefully I can continue in this tradition, periodically writing about the more amusing side of something that I find interesting and important. I will also, when I get round to it, post links to previous ramblings.

New Antibodies and New Data

We release several new antibodies. These are a new mouse antibody to muscleblind like protein 1, MCA-1H1, and several to Calbindin, made in both mouse MCA-5A9, MCA-4H7 and chicken CPCA-Calb (page in preparation). We also continue to develop a better understanding of all of our current reagents. For example, we previously made antibodies to the abundant cytoplasmic enzyme Aldolase C, both monoclonal and polyclonal. A potential problem with antibodies to this protein is cross-reactivity with the Aldolase A and B molecules which are very similar in amino acid sequence. Each Aldolase is 70-80% identical to the other two and the molecules have very similar molecular sizes on SDS-PAGE gels so one cannot distinguish which protein is being detected using western blotting. So we went to the trouble of expressing both human Aldolase A and B in order to test our panel of Aldolase C antibodies. We found that two of our monoclonals MCA-1A1 and MCA-4A9 were absolutely specific for Aldolase C, while a third MCA-E9 recognized all three Aldolases. We suspected that this might be the case since MCA-1A1 and MCA-4A9 were made against the C and N-terminal peptides respectively, which are the least conserved parts of the three Aldolases. In contrast, MCA-E9 was made against the intact human recombinant protein, and we now know that the epitope does not include either the N or C terminal sequences. Our rabbit polyclonal antibody to Aldolase C, RPCA-AldC, (page in preparation) not surprisingly, also recognizes all three Aldolase enzymes. Since Aldolases are abundant enzymes found in many tissues, we believe that using antibodies such as MCA-E9 and RPCA-AldC, which recognize all three, might be particularly useful as a western blotting standard, similar to our pan-specific actin antibody MCA-5J11.

News and Brain Cell Images

We went to the Society for Neuroscience 2014 meeting in Washington DC a few weeks ago, and presented our new antibodies, ELISAs, recombinant proteins and other products, which generated some interest. However the most interest seemed to be directed at our collection of high resolution images which we presented as 24″X36″ posters. We sold some of these and we had mentioned that we could also print these for use as Christmas wrapping paper. Well, we did this and the paper looks very nice, almost as nice as the posters. So we are now offering these for sale at reasonable prices. One sheet of 20″ X 29″ paper is $3.50 five is $14.00 and ten is $25.00. Shipping will be $6.00, irrespective of the number you order. We currently have two of these available, identical to our posters Image-2 and Image-7. We realize that it is too late for Christmas this year, but there is always next year, birthdays etc. The images are of high enough quality, despite the price, to make nice posters or wall decorations. We don’t have these two items in our on-line shopping cart yet, so just email us at admin@encorbio.com if you are interested in these.

Science News- STED microscopy

This years Nobel prize in Chemistry is a neat one, for the development of stimulated emission depletion (STED) microscopy. This is basically a modification of the existing confocal fluorescence microscope. Instead of scanning a specimen with a single laser to stimulate a fluorochrome the field is scanned with two lasers simultaneously. The first is the typical laser using the appropriate wavelength to excite a particular flourochrome. The second laser is the key new component, this is focused so that it produces a donut of light overlapping the focal point of the first laser. The wavelength of this laser is chosen so that it will prevent fluorescence emission at the normal emission frequency in the donut around the focal point. This works by the “stimulated emission” of the fluorochrome in the donut region, which inhibits emission at the normal wavelength, resulting instead in emission at a different wavelength which can be ignored by the microscope detection system. The end result is that the area from which the fluorescence signal is released becomes much smaller since the only region stimulated in the normal fashion is the small hole inside the donut. This region can be made significantly smaller than the region stimulated either in a regular confocal or in a 2-photon microscope. The end result is superior resolution, down to about 50 nanometers, about 4 times as good as either confocal or 2-photon microscopy. The technique can be used with two fluorochromes, so double labeling is possible, and the fluorochromes can in principle be any of the fluorescent probes in use today, either genetically encoded such as GFP or chemically coupled such as appropriately labeled primary and secondary antibodies (fortunately for EnCor, but see below). Unfortunately the only currently commercially available STED microscope systems are from Leica, not usually known for the marketing of inexpensive products. Even more unfortunately, Leica have an exclusive license on the patented STED technology, so no other company will be able to sell a STED microscope system for several years. This leaves the already embattled researcher with three options; 1. Beg, borrow, steal or in the absolute worst case buy a Leica STED system. 2. Build your own system, but not many people would know how to do that. 3. Don’t do anything but watch people who did 1 or 2 have all the fun. Here is a Leica STED tutorial so all you taking option 3 know what you are missing.

New Recombinant Human Proteins and Antibodies

We have had a long time interest in the intermediate or 10nm filaments of the nervous system, antibodies to which are very useful cell type and developmental markers. There has also been considerable recent interest in the detection of these proteins as biomarkers of different kinds of CNS damage and disease. In the last few weeks we have manufactured large amounts of several of these proteins in recombinant form. These will be used as ELISA protein standards, for monoclonal antibody epitope mapping and for continued production of more antibodies. All of the proteins were made using the pET expression system, and were expressed in and purified from E. coli.  All are expressed from cDNAs encoding the full length human protein sequence, but were codon optimized for efficient E. coli, expression. We currently have neurofilament NF-L (catalog entry Prot-r-NF-L), vimentin (catalog entry Prot-r-Vim), peripherin (catalog entry Prot-r-Peri), GFAP (catalog entry Prot-r-GFAP) and α-internexin (catalog entry Prot-r-a-Int), and are producing other similar constructs on a routine basis, including those related to general neuroscience. We also introduce new monoclonal antibodies to GAP43, MCA-5E8 and MCA-3H14 and a new monoclonal antibody to microtuble associated protein tau MCA-5B10.

Aurora A, B and C antibodies

A few months ago we made some mouse monoclonal antibodies to the interesting cell division and cancer related Aurora A, a highly conserved serine/threonine protein kinase. These seemed to work very well but we subsequently found that some of them cross-reacted with the very closely related Aurora B. We therefore went to the trouble of expressing all three members of the human Aurora kinase family, Aurora A, B and C, and we now have mouse monoclonals which are monospecific for each individual Aurora family member (Aurora A = MCA-1A11, MCA-1A14, Aurora B = MCA-3F11, MCA-6G2, Aurora C = in progress, will update on that shortly). We also have a pan-specific mouse monoclonal antibody which will bind all three Aurora proteins (MCA-4A7) and two monoclonals which bind Aurora A and B but not C (MCA-5A12, MCA-3H1). All members of this panel of novel antibodies work very well on western blots of crude extracts of HeLa cells and in immunocytochemistry on cells in tissue culture. They should therefore be useful as markers of midbodies, spindles and polar bodies in dividing cells and for developmental and cancer related studies.

The New Web Site again…

We we have certainly had some problems with the new website, but not as much as Health.gov, as a few customers did actually manage to navigate the system well enough to successfully buy things. We are on this though and will be working hard to get everything working smoothly. One cool thing is our slideshow here, which you can maximize and run as a perpetual screen saver if you want. Since we thought that was pretty cool we will be looking for older images which look good in that kind of format from now on. In the next post we will describe several new products which we did not get to describing due to the pain and suffering of getting the new site up. Have great weekends! (One for each of you).

The New Web Site…

Well the new web site is more or less working now, though we still have to iron out quite a few little problems. If you are having trouble ordering anything, just call 352 372 7022, fax 352 372 7066, email to admin@encorbio.com or use our older on line order forms for purchase orders at here, and for credit cards here. Or download a pdf version of our form here.

New Antibodies

We announce two new mouse monoclonal antibodies to tubulin, the major protein of microtubules and one of the major proteins of mammalian cells. These antibodies are excellent for visualizing microtubules in cells in tissue culture. They are also very useful western blotting standards, producing extremely strong and clean signals. Details will be posted in the next few days.

Welcome to EnCor’s new web portal

We are finally ready to post our new web site, which includes all of the features of our previous site and also an online store. You can now conveniently browse our various antibodies, ELISA kits, protocols and images.