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February 2015 Archive

Posted by Karen on February 25th, 2015  ⟩  2 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.

how to choose between similar reagents

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 Karen on February 17th, 2015  ⟩  0 comments

As children, we’re often taught complex subjects in very simplified ways. Life was easier in black and white, but as we move into adulthood, we find that the world is far more colorful than that.

Even as young adults, we learn that a basic experimental setup should have a control and a single independent variable to test. But the world is not the vacuum our textbooks taught us, and in our experiments, we must consider other variables that interfere with our research, which often go severely undetected.

One element of interference is contamination. Thankfully, in lab classrooms, we’re taught sterile methods right away, and we develop a “when in doubt, throw it out” philosophy with our pipette tips, gloves, tubes and, as much as it kills us, our samples.

In the field of plant research, specifically in agronomical crops like rice and corn, Norman Best and a research team from Purdue University and Chicago State University investigated another avenue for interference: unfavorable biochemical reactions in plant media.

In experiments requiring the study of larger plants, the neutral Murashige and Skoog medium is not as efficient or affordable. Instead, researchers use soilless growth substrates such as sphagnum peat moss and calcined clay. Other additives such as perlite or vermiculite are also included. Each of these products are chosen for different reasons and have unique characteristics that optimize growth in certain ways. Peat moss retains large amounts of water within the cells of live and dead plants, and it can retain water up to 60% of its volume. Though water retention is a benefit from this medium, it is not very nutrient rich and the soil tends to become more compacted. It also has a pH of 3.0-4.0, while optimal levels are usually around 5.5-7.0.Perlite is often used in potting soil, and has also become a widely used medium in hydroponics. This is because it is known for having greater permeability. Since the material is derived from volcanic rock, it holds its structure over time, allowing the long lasting aeration. It is also has a pH of 7.5. The downside to perlite is that it has lower water retention, and it requires nutrient rich water to nourish plants. Vermiculite is used similarly to perlite, in that it’s meant to condition the soil for proper aeration. While it does retain slightly more water than perlite, it does not hold its structure for very long. Therefore, it is not good for outdoor growth or hydroponics. The most popular growth medium for larger plants, however, is calcined clay because nutrients are more slowly release. Clays like Turface®, which are used in baseball fields, can absorb its weight in water, keeping water levels at an optimal amount.

While people tend to choose a specific medium for its ability to facilitate growth, Best examined interactions between soilless growth media and various plant hormones such as brassinosteroid and its inhibitor propiconazole.

Brassinosteroids, also called BRs, are plant hormones involved in a number of activities. Most notably, they promote stem elongation, cell division, pollen elongation and stress protection. Mutants lacking proper levels of BRs demonstrate lower seed germination, flowering delays and dwarfism. Therefore, inhibitors such as propiconazole allow scientists to better understand molecular pathways involving this hormone.

Along with the brassinosteroid inhibitors, Best also gained more insight into reactivity with the plant growth media using GoldBio’s GA3, and its inhibitor uniconazole. Gibberellic acid stimulates cell elongation and excites the cells of germinating seeds. Its inhibitor, uniconazole or Ucz, is an ingredient in plant growth retardants, and is often very active in small concentrations.

Through his experiments, he observed significant interactions between the plant growth regulators and the growth substrates, which greatly reduced the inhibitors’ efficacy. This was particularly observed in calcined clay, which was attributed to hydrophobic interactions between the plant growth regulators and growth media.

While calcined clay is often preferred, its heavy interaction between plant growth regulators may decrease experimental optimization. However, vermiculite showed much lower chemical reactions with the plant growth regulators. And, the results showed that there was very little interaction between GA3 and soilless growth substrates, which might be reassuring to researchers who use the hormone.

The analysis of this study provides researchers a new criterion to be considered when choosing the appropriate growth media. For basic gardening, using growth media based on its ability to hold water or nutrients is appropriate. However, when studying molecular pathways, it becomes even more important to understand new elements of interference which may have an undesired influence on the result.

Though we may at times be oblivious to the shades of green of hidden variables within our experiment, the results from Best and his team have highlighted one more variable, allowing subsequent research to become slightly better controlled as a result.



Best, N., Hartwig, T., Budka, J., Bishop, B., Brown, E., Potluri, D., Cooper, B., Premachandra, G., Johnston, C., Shulz, B. (2014). Soilless plant growth media influence the efficacy of phytohormones and phytohormone inhibitors. Plos One. Doi: 10.1371/journal.pone.0107689


    
              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: 88241