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DTE

Posted by Karen on February 25th, 2015  ⟩  0 comments

There are a lot of products in the lab that do similar things but were developed for a specialized purpose. And sometimes you’re in a position where only the alternative is available, but you’re unsure whether you can trust it.

Let’s take a look at a few of these products and see exactly what the difference is in order to determine which one is more appropriate, given a certain experiment.

1. Tris vs. Tris HCl

     vs.  

Both can be used for electrophoresis, but why would you choose one or the other? Tris is much less expensive compared to tris HCl; however, tris HCl is meant to simplify the buffer-making process.

On its own, tris is the basic component of the buffer, while the acidic component of the buffer would come from adding HCl. Rather than working with HCl or NaOH (to adjust the pH of tris HCl), tris and tris HCl can be blended together to reach the desired pH.

Making a tris buffer solution with tris-HCl also prevents overshoot, which occurs when too much acid is accidentally added, meaning NaOH will need to be added to correct the situation.

Verdict:
In the end, it’s handy to have both in the lab. But either or can be used, so long as you have the acid or base to properly adjust it.

2. XTT vs. MTT

          vs.      

Once again, the most obvious difference is price. XTT is priced higher than MTT, yet they are both very similar in application. So how do they differ? And what justifies the difference in price point?

XTT holds some significant advantages over MTT. For example, XTT is very sensitive and has a higher dynamic range. Reactions with XTT result in a soluble formazan dye. This means the final solubilizing step is eliminated, which is not the case when using MTT. Removing this step also reduces the risk of error, such as air bubbles from Triton X-100 or SDS. Lastly, OD reading can be immediately taken after incubation when using XTT, while MTT usually requires a longer incubation for the solubilization of precipitate.

Verdict:
Whether you choose XTT or MTT, the job will get done. Choosing between the two depends on your preference. If you want to eliminate steps and reduce the potential for error, then XTT is your guy. But if those advantages are not worth the additional cost, which is very significant, then MTT is a great alternative. Both are very reliable for evaluating cell populations.

3. DTT vs. β-mercaptoethanol vs. TCEP HCl

      vs.          vs.     

If you have worked with β-mercaptoethanol (βME) in the past, then you are too familiar with the stench. Even after you dispose of your gloves, the smell lingers in your nose for some time. Despite its unforgettable scent and toxicity, it is much less expensive than DTT or TCEP. Sometimes saving money is worth the obstacles; however, there are some preferred applications for each of the three.

TCEP is known for its stability and lack of odor. According to some posts on Research Gate, many researchers prefer to use it for storage. By only storing protein stock in TCEP, you use less and incur a lower expense. TCEP is also useful if you’re doing UV detection of protein in buffer. This is because TCEP absorbs less UV than the other two reagents.

During protein purification, βME or DTT are the popular choices. DTT is a strong reducer, 7-fold stronger than βME, and it doesn’t have the odor that comes with βME. On the other hand, βME is far less stable. It evaporates from solution which means its concentration in solution will decrease with time. And to maintain equilibrium, more βME is required; otherwise, proteins won’t be sufficiently reduced, causing electrophoretic bands to be fuzzy.

Ultimately, it’s a matter of preference between the two, however some researchers suggest there being a benefit to using DTT for protein purification and for using βME when purifying smaller molecules.

Verdict:
In the end, this is yet another series where all candidates should have a place in your lab. Though if you must boil it down to two, choose DTT and TCEP. Neither of the two products smell quite as bad, and GoldBio’s prices justify the subtle advantages.

4. DTT vs. DTE

     vs.   

With all of this talk about reducing agents in the previous example, you might now be wondering about DTT vs. DTE (Cleland’s Reagents). This is one case where it’s simply a matter of preference and price. In Cleland’s original article, he stated that there appears to be no significant difference. They are epimers: the hydroxyl groups of DTE are in the cis form, while in DTT, they’re in the trans form.

Verdict:
Either or is fine. Go with the more cost effective product or the product you have the most experience with. At least now you know that if you’re ever in a situation where you must “borrow a cup of sugar” from another lab, either product you’re given will work just fine.

5. Ampicillin (Sodium) vs. Carbenicillin (Disodium)

    vs.     

Ampicillin is widely used for selection during cloning experiments; however it has its drawbacks. Carbenicillin is a more stable substitute, but like the previous examples, advantages always come with a cost. So do the benefits of carbenicillin outweigh that cost? And, are there times where one antibiotic is more advantageous than the other?

During selection, cells containing the bla gene from transformation will show resistance to ampicillin by expressing beta-lactamase, which inactivates ampicillin. The problem is that beta-lactamase is secreted by the bacterial cells, and when enough extracellular accumulation occurs, ampicillin in culture can be inactivated. What this ultimately means is that you can have a lower yield of desired cells in liquid culture, and satellite colonies may appear on agar plates (“These aren’t the cells you’re looking for”). As your plates age, the risk for satellite colonies increases.

Carbenicillin is more stable than ampicillin. It has a higher resistance to heat, it won’t degrade as easily in a lower pH, and it has an increased shelf life. But the other benefit is that satellite colonies are less likely to form since carbenicillin lasts longer and is less susceptible to hydrolysis by beta-lactamase. While all that sounds good, the risk when using carbenicillin is that its potency may kill cells before they have time to manufacture the resistance.

Verdict:
Keep both in the lab, and know when to use one over the other. When you’re dealing with quantification and longer incubation times, it’s safer to use carbenicillin. But when you’re doing a ligation, your cells are slow growing or your DNA is fragile, it’s safer to use ampicillin.

So there you have it, a starter guide for some common reagents that do very similar things. We know this is a list of only five categories. So if you think we need to have a part two, please chime in with your suggestions, questions or even your answers about other products!


    
              Karen Martin
GoldBio Marketing Coordinator


"To understand the universe is to understand math." My 8th grade
math teacher's quote meant nothing to me at the time. Then came
college, and the revelation that the adults in my past were right all
along. But since math feels less tangible, I fell for biology and have
found pure happiness behind my desk at GoldBio, learning, writing
and loving everything science. 



Category Code: 88253 79108 79107

Posted by Chris on January 10th, 2013  ⟩  0 comments

Previously, we’ve been discussing the function of various reducing agents. There are a number of reducing agents that are available at Gold Bio: DTT (dithiothreitol), DTE (dithioerythritol), L-glutathione (GSH) and TCEP (Tris (2-Carboxyethyl) phosphine hydrochloride). By definition, reducing agents are elements or compounds that donate an electron to an oxidizer compound. The agent of the day is DTE (dithioerythritol).

DTE is an epimer of DTT (dithiothreitol), where the two hydroxyl groups of the oxidized, disulfide-bond form of the compound are in the cis form (rather than the trans form of DTT). Traditionally both have been called Cleland’s reagents synonymously. DTE is a strong reducer for the same reasons as DTT; the stable oxidized ring structure (with its internal disulfide bond) makes it hard to oxidize back into its reduced form and easier to complete the reduction of the disulfide bonds of a protein. DTE is considered to have a slightly lower reduction potential than its sister compound because of the cis state of its hydroxyl groups. But it is still considered to be one of the best reducing agents available!

DTE has been utilized in a wide variety of research over the years and cited in literally thousands of articles since its initial discovery by Cleland in 1964. From 2-D electrophoretic analysis of proteins and immunoglobulins, research into glial and neuronal transport, the glycation of hemoglobin in GSH-deficient red blood cells, to the more recent determination of steroidal hormones in saliva samples. DTE and the other reducing compounds sold by Gold Bio continue to be crucial reagents in scientific discovery and experimentation. For instance, Alessandro Cavallo et al. are using it to determine the CPT-I (Carnitine palmitoyltransferase–I) activity in rat liver tissue. CPT-I is the primary enzyme in control of fatty acid oxidation and the hepatic mitochondrial β-oxidation flux. Their work may one day elucidate the roles such enzymes play in response to critical thyroid hormones in the development of liver steatosis and rat adiposity.

 
 

Fialka, Irene, et al. "Subcellular fractionation of polarized epithelial cells and identification of organelle‐specific proteins by two‐dimensional gel electrophoresis." Electrophoresis 18.14 (1997): 2582-2590.

Layer, Andréas, et al. "Micropurification and two-dimensional polyacrylamide gel electrophoresis of immunoglobulins for studying the clonal diversity of antigen-specific antibodies." Journal of immunological methods 227.1 (1999): 137-148.

Jain, Sushil K. "Glutathione and glucose-6-phosphate dehydrogenase deficiency can increase protein glycosylation." Free Radical Biology and Medicine 24.1 (1998): 197-201.

Oh, Jin-Aa, and Ho-Sang Shin. "Rapid determination of natural steroidal hormones in saliva for the clinical diagnoses." Chemistry Central Journal 6.1 (2012): 22.

Cavallo, Alessandro, et al. "3, 5-Diiodo-L-Thyronine Administration to Hypothyroid Rats Rapidly Enhances Fatty Acid Oxidation Rate and Bioenergetic Parameters in Liver Cells." PLOS ONE 8.1 (2013): e52328.

Category Code: 88221 88251 55123