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February 2013 Archive

Posted by Chris on February 13th, 2013  ⟩  1 comments

February 14th is celebrated as Valentine’s Day in the US. We mark the occasion by collectively giving flowers, chocolates, dinners and gifts to the special people in our lives. News agencies detail that American consumers dish out over $13 BILLION in various ways on Valentine’s Day alone, over $3 Billion on just flowers and chocolate! That’s a lot of chocolate!

There has always been a lot of news fodder concerning chocolate and whether it’s good for you or bad for your health. Researchers have pointed to numerous chemicals inherent in the cocoa plant (the source of all things chocolate) as well as the processed chocolate that most of us actually eat. They’ve detailed research article after article on the detriments of the sugars and caffeine which we are all well aware of, or the benefit of other chocolate chemicals such as theobromine, a mild natural stimulant similar to caffeine, or 2-phenylethylamine (PEA), an neurotransmitter type drug which may or may not be responsible for that pleasurable feeling you get after eating your twelfth bonbon.

The most recent double-blind studies have shown that, contrary to popular hope, theobromine (and therefore chocolate) doesn’t actually lower blood pressure or significantly improve your mood. And while PEA may do wonderful things to test animals when delivered directly into the brain, when ingested it is quickly degraded into phenylacetic acid which is terrifically inactive in the body and thus removes almost any possibility that PEA would ever actually cross the blood brain barrier to fill us full of endorphins.

But wait! Before you ditch all of the Valentine traditions, did you realize that roses are actually quite good for you? Roses are by far the most plentiful flower presented on Valentine’s Day, and 71% of men and 36% of women purchase flowers for the occasion. The aggregate fruit of the rose blossom, called a “rose hip”, is actually a treasure-trove of vitamins and nutrients (though over-domesticated cultivars might not develop hips for lack of access to pollination). The rose hip is one of the richest sources of Vitamin C in plants (they also have Vitamin A and B)! HPLC assays have shown them to have a wide range of L-ascorbic acid, a naturally occurring antioxidant. They contain lycopene a type of caretene typically found in vegetables like carrots or tomatoes, which also has a lot of antioxidant activity and is a key component of LDL’s (low density lipoproteins). And rose hips have been found to have anti-inflammatory properties and may be useful in treating rheumatoid arthritis. That's not a bad resume for a flower that typically just meant to represent your love for someone.

GoldBio wants to wish you a wonderful Valentine’s Day and remember: if you want to have a treat or two of something healthy this year, just go ahead and nibble on the roses!


 
 

van den Bogaard, Bas, et al. "Effects on Peripheral and Central Blood Pressure of Cocoa With Natural or High-Dose Theobromine A Randomized, Double-Blind Crossover Trial." Hypertension 56.5 (2010): 839-846.

Mitchell, E. S., et al. "Differential contributions of theobromine and caffeine on mood, psychomotor performance and blood pressure." Physiology & behavior 104.5 (2011): 816-822.

Shulgin, Alexander, and Ann Shulgin. "PiHKAL." Transform Press, Berkeley, CA (1991).

Rodriguez-Amaya, Delia B. "A guide to carotenoid analysis in foods." A guide to carotenoid analysis in foods 64 (1999).

Larsen, Erik, et al. "An Antiinflammatory Galactolipid from Rose Hip (Rosa c anina) that Inhibits Chemotaxis of Human Peripheral Blood Neutrophils in Vitro." Journal of natural products 66.7 (2003): 994-995.

Category Code: 88221 79101

Posted by Chris on February 7th, 2013  ⟩  0 comments

There are a number of great methods in the researcher’s toolbox for protein purification. Along with the ever popular His-Tagged protein purification, there is also the choice of GST-tagged proteins. Glutathione S-transferase is a super family of enzymes that have been utilized by researchers to create the GST gene fusion system. Due to GST’s high affinity for reduced glutathione, it is incredibly easy to pull the GST fusion protein (combined with your protein of interest) from a cell sample. For that purpose, many researchers use a glutathione crosslinked agarose resin as a matrix for binding to the GST fusion proteins. A simple rinse with a reduced glutathione solution and a pure protein sample is yours!

Of course, there are always some limitations. The GST tag is comparatively large, around 26 kDa (the His-tag is around 1 kDa), though it is not as large as Protein A (30 kDa) or the Maltose binding protein (40 kDa). But many commercially available GST tags usually contain a convenient cleavage domain which can then be used to remove the purification tag with a great deal of specificity (just make certain you don’t have a similar site on your protein of interest!). It is also almost always fused to the N-terminus of the protein, which may limit your ability to purify it depending on your protein’s structural configuration. But generally, proteins are stably folded when fused with the GST tag. It has further advantages in stabilizing small or unfolded protein sequences! And there are also many commercially available anti-GST antibodies for use in downstream assays involving your GST tagged protein.

GST is also very susceptible to denaturing, leading GST-tagged proteins to display a ladder of lower MW bands after an SDS-Page protein gel. In fact, purification of a GST fusion is all but impossible in the presence of even very low concentrations of SDS. But what if your protein has to be solubilized in SDS? Elodie Boisselier et al. developed a very interesting strategy to do just that. They found that the addition of 2-methyl-2, 4-pentanediol (MPD) to the protein mixture before running it through a column containing SDS proved sufficient to blocking the adverse effect of the SDS on the GST protein!

Regardless of your specific protein research needs, our goal at Gold Bio is to continue to support you with the quality reagents (and tips!) you might need to get the job done. And if you have any questions, please contact us at techsupport@goldbio.com.

 
 

Douglas, Kenneth T. "Mechanism of action of glutathione-dependent enzymes." Adv Enzymol Relat Areas Mol Biol 59 (1987): 103-167.

Oakley, Aaron. "Glutathione transferases: a structural perspective." Drug metabolism reviews 43.2 (2011): 138-151.

Production of HexaHistidine or Glutathione-S-transferase (GST) tagged proteins in Escherichia coli (Ponnambalam Lab, Leeds)

Elodie Boisselier, et al. “A strategy for purifying glutathione S-transferase in the presence of sodium dodecyl sulfate.” BioTechniques 51.3 (2011): 193–194

Category Code: 88221 79105 88253

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 Patrick on February 25th, 2013  ⟩  0 comments

To coincide with the introduction of our new Protein G antibody purification resin, we thought that we’d do a quick introduction to go over the steps and considerations you may need for antibody purification.  Purified antibodies can be a powerful research tool, but the steps necessary to purify them correctly and efficiently can be pretty tricky to nail down without a good amount of knowledge, and some hard work. Hopefully these recommendations will help set you on your way to getting that purified antibody you’ve been waiting for.

The first step in making sure your purification is on the right track is to make sure that you’re using the correct purification method. Depending on your final use, you may not need a highly purified antibody, and may be able to just use the tissue culture supernatant or antiserum if your assay isn’t concentration dependent. If you do require a more purified antibody, then an affinity purification is one of the fastest and easiest ways to filter the total immunoglobulin amounts out of different lysates or media.

In order to get the best yield from your affinity purification, it’s important to match the class of your desired antibody to the purification resin you plan to use. For example, if the antibody you’re attempting to purify is a monoclonal Rat IgG2a you would want to use a Protein G resin, due to its much higher ability to bind that subclass of IgG than Protein A. Alternatively, if you’re attempting to purify a polyclonal Rabbit antibody, you might want to choose a Protein A resin due to its slightly higher binding ability. If you are unsure about which resin would be more effective for your needs, most manufacturers will sell a test kit of their different resins in order for you to test them out for yourself.

Once you have the correct resin you want to use, it’s important to carefully go over the purification process. Depending on the sample you are trying to purify, it may need to be filtered or centrifuged to remove any excess particulate matter that may decrease binding ability or clot the column during purification. A correct pH is also essential for the antibody to bind to the Protein A/G resin, so it’s very important that the sample is pH’d before adding to the column. Typically for binding , a pH of 8.0 is used, but some research shows that certain subclasses bind better at a lower pH.  Elution conditions are also very pH dependent, as the antibody will only dissociate from the Protein A/G if the pH is very low, typically 2.8. As the antibodies will be pretty unstable at such a low pH, after purification you will need to either preform a buffer exchange or neutralize the eluate with 1M Tris-HCl (pH 8.5).

Hopefully these steps will help get your research on the right track. There are a lot of great resources out there to help you continue your research with your purified antibody, and soon you’ll be able to harness all the ability that purified antibodies can offer. Please contact us with any questions at techsupport@goldbio.com.

Category Code: 88253 79103 79107

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