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Tagged with “protein purification”

Posted by Chris on February 13th, 2014  ⟩  0 comments

In the comfortable culture of the U.S., which we take for granted all too often, it can be difficult to remember the human plagues and diseases that have followed mankind throughout the millennia. It’s difficult to remember that we, ourselves, are the preferred breeding ground for a host of bugs, worms and parasites; not just idle or incidental carriers of these microscopic beasts, but their main course…dependent on our unique physiology in order to reproduce and survive.

Some of these diseases are famous, due to their pugnacious nature or severity of effect; diseases such as malaria, botulism, or River Blindness. Others are less well known, either due to their more limited environment or because their incidence in more developed countries is less common, such as the protozoan, Entamoeba histolytica. However, less common does not necessarily mean less destructive. Entamoeba is a group of anaerobic parasites that specifically target humans and primates. Typically passed from feces and water to another host in a cystic form, these protozoans mature in our digestive tracts and cause a disease called Amebiasis. Amebiasis is typically characterized by abdominal pain, amebic dysentery, bloody diarrhea, and fevers. The protozoans can also occasionally escape the colon and end up on other organs to cause liver, brain or lung abscesses. Current estimates are that nearly 50 million people, worldwide, suffer infection from E. histolytica, which result in around 100,000 deaths. However, only 10-20% of all infections become symptomatic, so the number of infections may actually be much higher. The rate of infection of E. histolytica in tropical countries in Central or South America, Africa and Asia is actually nearer to 50%! And when the numbers of E. histolytica are combined with similar, but non-symptomatic protozoans (such as E. dispar and E. moshkovskii), the total world count may be closer to 10% or 500 million infective cases.

Protozoan immunofluroescentThe most likely place to start looking for better methods of disease prevention of Amebiasis are the surface protein interactions between the protozoan and our intestinal walls which cause adherence and activation of the protozoan to its trophozoite stage. Cataloguing and defining these surface proteins is about as easy as identifying all of the various countries on Earth from a telescope on Mars. To date, only a smattering of about 20 of these surface proteins had been identified. Iris Bruchhaus and her group from the Nocht Institute for Tropical Disease wanted to blow that number away and settle once and for all what proteins need to be studied.

Bruchhaus’ group used a non-permeable biotinylation process (similar to Gold Bio’s Biotinylation kits found here) to bind with all of the surface proteins on the HM1:IMSS strain of E. histolytica. Those biotin conjugated proteins were then easily isolated using a streptavidin agarose resin system (which you can also conveniently find here), and analyzed in NanoLC-MS/MS in order to identify the proteins. They found close to 700 proteins! Oddly though, nearly 50% of the proteins found they found did not show any specific membrane association. Bruchhaus’ group was able to further detail many of these isolated oddball proteins, showing that roughly 85% of them did have some cell surface interaction, even if not in the traditional sense. But this actually implies that the plasma membranes (and their surface associations) of these protozoans are not static and easily defined at all, but are a very dynamic, complex and interconnected weaving of molecules in constant exchange on and across the membrane.

That doesn’t necessarily make this type of work any easier. But with a large number these proteins identified, work can at least begin on some of the most important proteins, further characterizing the association with their human host cells. Who knows, maybe it’s actually one of these oddball proteins that only sometimes associates with the plasma membrane that account for the low percentage of the protozoan becoming symptomatic. Time will tell…

 
 

Biller, L., Matthiesen, J., Kühne, V., Lotter, H., Handal, G., Nozaki, T., Saito-Nakano, Y., Schumann, M., Roeder, T., Roeder, E., Krause, E., & Bruchhaus, I. (2014). The Cell Surface Proteome of Entamoeba histolytica. Molecular & Cellular Proteomics, 13(1), 132-144.

Category Code: 88241 88231

Posted by Chris on February 7th, 2014  ⟩  0 comments

Apparently, I am a dog person. It might actually be truer to say that I like all animals, but I’m sure that if you were able to ask my dog her opinion, she would tell you: I am a dog person. As a product of my culture, I spend an inordinate amount of money and time on my dog. Studies show that Americans spent over $60 Billion on their pets in 2013, with slightly more than half of that on just their dogs alone. Specifically, nearly 46 million households have provided a comfortable, all-inclusive, resort-like home for nearly 80 million dogs in the United States in 2013. Those trends have been steadily increasing for years and do not look to taper off anytime soon.

As we improve the quality of life for our dogs, their life expectancy has increased, just like ours did in the throughout the 20th century. It’s no surprise, then, that dogs are seeing an increase in cancer rates, partially due to their extended life expectancies…again, similar to our own history of complications with increased life expectancy. One of the most common forms of bone cancer in dogs is called Osteosarcoma or OSA. Its prevalence most often strikes in larger, and older, dogs and accounts for about 85% of canine skeletal tumors. OSA CancerOSA occurs often around the leg joints and treatment usually involves amputation followed by chemotherapy. However, even with treatment, the prognosis is very poor. Approximately 90% of dogs succumb to metastasis and the average life expectancy, post treatment, is only 6 months. Tragically, there is just a 50% chance of surviving a year and but a 25% chance of our best friends surviving beyond that point.

As with all cancers, the key to survival is earlier detection and earlier treatment/therapy. But it’s not as if our beloved pets can tell us when something hurts or just doesn't feel right. More often than not, they play through the pain, ignoring all but the worst of their injuries and ailments. And let’s face it, most of us pay less attention to the small clues they do give us than we should, further complicating the job of our veterinarians. To help address this tragic problem, a group from the College of Veterinary Medicine in Oregon, began the introductory process of identifying and defining the surface-exposed proteins (SEP) of OSA cancerous cells.

Milan Milovancev et al. used a biotinylation/streptavidin enrichment process to label and identify with Mass Spectrometry more than 100 putative SEP candidates from verified OSA cell lines as compared to normal osteoblasts. Utilizing the strength of the biotin-streptavidin association, Milovancev’s group biotinylated the surface-exposed proteins in the cells (for a similar system, see Gold Bio’s Biotin conjugation kits), lysed the cells to release the proteins and separated the biotinylated proteins using streptavidin-labeled beads…simple, clean and efficient.

Of course the enrichment process, by its own method, skews the quantification of the protein levels in the tumor cells. But the purpose of this study was only to find putative candidates, which can then be further studied in depth at a later time. To that end, Milovancev’s group set out to validate these initial results with secondary identification methods, such as RT-PCR, Western Blots and Immunocytochemistry. The follow up results mostly (but not completely) verified the results of the enrichment/MS identification method, meaning that while the system will do what it’s supposed to in general, Milovancev is just going to work harder to achieve a future, better refinement of the procedure.

As a dog person, I (and my dog) wish him well.

 
 

Milovancev, M., Hilgart-Martiszus, I., McNamara, M. J., Goodall, C. P., Seguin, B., Bracha, S., & Wickramasekara, S. I. (2013). Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts. BMC Veterinary Research, 9, 116.

Category Code: 88241 88231

Posted by Chris on February 28th, 2013  ⟩  0 comments

Chances are that if your research involves affinity chromatography, then you’re probably already using or have previously used Cyanogen Bromide-activated (CNBr-activated) agarose beads to bind or purify your proteins, antibodies or enzymes. That’s the industry standard for ligand immobilization! It’s great! It works! It’s wonderful!

But what if Gold Bio could offer you something better...and less expensive?

CNBr is often used to immobilize proteins by coupling with agarose beads. Usually, the agarose is either 4% or 6% (which typically sets the exclusion limit of the beads) and can be crosslinked or not (crosslinking permanently binds the linker to the agarose which offers the advantage of autoclaving the beads and/or re-using them). CNBr is used so often because it reacts simply and easily with the hydroxyl groups on agarose to form cyanate esters and imidocarbonates, which then react with the amino group of proteins, binding them to the agarose. But CNBr is quite toxic and it’s also very sensitive to oxidation. And its ligand binding is relatively unstable because of its isourea bond which tends to act as an anion exchanger.

At Gold Bio, we want to provide you with the best reagents possible for your research. To that end, we offer two alternatives to the classic CNBr resin: Glyoxal- and Aminoethyl-activated agarose beads!

Our Glyoxal/Aminoethyl agarose beads are all crosslinked, so that you can autoclave them and re-use them multiple times. They are covalently bound to the agarose, giving them a qualitative advantage to resins activated by CNBr. They have a higher binding capacity, faster conjugation, more reproducibility and a longer shelf life than CNBr. You can find a comparison study of Human IgG binding between Glyoxal and CNBr agarose in the Additional Information of the products in the Glyoxal section or Aminoethyl section of our website.

For another comparison of binding capacity, check out this chart below:

The combination of Glyoxal/Aminoethyl system offers more versatility for your research. While you would use the Glyoxal resin to bind with the amino group of your protein (similar to CNBr), you can use the Aminoethyl resin to bind with the carboxy group of your protein, an opportunity that CNBr resins cannot match!

You can find a detailed list of comparable Gold Bio products to some of the more common CNBr resins in the Additional Information of either the Glyoxal or Aminoethyl products. If you still have questions about these products or about which one you should try, please contact our tech support and we will be glad to help!

Category Code: 79105 88253 79107 88261

Posted by Chris on February 21st, 2013  ⟩  0 comments

One of the products that you’ve repeatedly asked us to carry at GoldBio is a Protein G Agarose resin for affinity purification. So we’re excited to tell you that we now have a reliable and cost-effective Protein G Agarose resin (Catalog # P-430) available for your research needs!

Protein G is a 23 kDa cell-surface protein from the group G Streptococcus species that is often used in the purification of antibodies because of its great affinity with the Fc (crystallizable fragment) region of immunoglobulin G.

Similar to Protein A, Protein G has been used to extensively to detect and purify IgG antibodies and antigen/antibody complexes, and it has efficacy over a wide pH range (pH4-8). But Protein G has a wider range of reactivity over more species than Protein A (including goats, rats and sheep). Most notably, Protein G binds to Human IgG3 and Mouse IgG1, two antibodies that Protein A reacts with poorly. You may find a more inclusive list of monoclonal and polyclonal antibodies associations in the additional information page for Protein G. One potential drawback of native Protein G is that it tends to bind to human serum albumin (HSA), though that does not seem to affect its ability to bind to Human IgG. However, GoldBio’s recombinant Protein G Agarose resin has been genetically engineered to remove the albumin-binding domain as well as the cell wall and cell membrane binding domains to ensure the maximum specific IgG binding capacity.

We hope that you are as excited as we are about this new product. We will continue to look for the best products for your research and the best prices for those products to pass along to you. And if there are any other products that you would love for us to carry, please contact us and let us know!

Category Code: 88261 88251

Posted by unknown on February 19th, 2013  ⟩  0 comments

Dear Readers,

I am excited to announce that we are launching the Goldbio Video Reference Library today!  As long as we've been selling our high-quality products, we've been hard at work in the lab using them too. Now we're turning our experience into something you can use. The library will contain original Goldbio-created content. Since demonstration is how we learn in the lab, videos give us the opportunity to provide instruction to anyone, anywhere, any time. Through these videos we plan to share tips, tricks and techniques along with protocols, product demonstrations, and answers to frequently asked technical questions. It is our way of sharing with you what we love to do - research and develop the tools needed push our industry farther. We believe that collaboration, communication, and demonstration of pertinent ideas and techniques will help lead to discovery and innovation - and that is what we are passionate about.

We are beginning with a video protocol for pouring and packing an agarose column with Patrick.  In the coming weeks we will have an additional protocol, answers to some frequently asked technical questions on IPTG, and a very special new product line announcement.  There is a place for you to contact us about any content you have questions on or would like to see us cover.  So talk to us, let us know what you think and what you’d like to see and we will do our best to get it to you. Our goal is to encourage interaction among researchers that will lead to better projects, improve life in the lab, broaden understanding, accelerate research and enable those breakthrough moments that benefit all. We sincerely hope that you will enjoy and share these videos with your colleagues, and visit our library often. You never know what great tips you'll pickup!

Sincerely,

Bart Saracino

Director of Marketing, Gold Biotechnology, Inc.

Category Code: 88261