Get To Know Your Common Reaction Reagents
When I first started out in the lab, I used to follow all protocols to the letter. Now, this is fine as long as your reactions run smoothly. However, there came the day when I had a new protocol and I just couldn’t get it to work. My supervisor told me to just “play around” with the parameters until I got results.
Easier said than done, for I had an idea of how the reaction worked in theory, but I had no clue what most of the reagents in my protocol were actually doing. What should I take out? What to add? I was completely lost.
Ever since, I made it a habit to look up all the compounds of a reaction that I don’t know yet. So if you are anything like me, only you never got around to checking out those chemicals or maybe you are just starting out in the lab, this list just might be of use to you.
DMSO (dimethyl sulfoxide)
DMSO started out its life as a by-product of the wood industry, but since then has become a widely used organic solvent. In the lab it can be found in many places such as part of PCR mixtures where it helps to unfold the secondary structure of the DNA and the primers. Here it facilitates annealing of the primers and elongation via the polymerase. In the same way DMSO can be added to reverse transcription mixes to help unfold RNA structure. A good alternative for DMSO in these cases is Betaine, which also helps unfold nucleic acids.
DMSO is also used in cell culture, where it serves as a cryoprotectant (sounds like something out of Star Wars doesn’t it?). It simply means that DMSO will protect the cells during the freezing process. The main danger when freezing cells is that water inside the cells will freeze and form ice crystals. Not only do these crystals have sharp tips that could injure the cell, but also water in its frozen form will take up more space than water in its liquid state. This means that the frozen water within the cell will expand and could ultimately explode the cell. Such disasters can be prevented by a cryoprotectant such as DMSO (or glycerol). The cryoprotectant will penetrate the cell and displace the water, thereby protecting the cell from freeze injury.
Whatever you use DMSO for, be aware that it can easily penetrate your skin. DMSO itself is not the problem, but it will take with it any chemical agent that it happens to be in contact with. This comes in handy for some medical uses, when you want a certain drug to penetrate into the cells. But in a lab there are lots of things that you do not want under your skin, so always use gloves when working with DMSO.
William Wallace Cleland was the first to describe the potential of DTT to reduce disulfide bonds in proteins (which is why DTT is sometimes still referred to as Cleland’s reagent). But why would one want to reduce disulfide bonds in the first place?
Many proteins (and even DNA) have free (unbound) sulfhydryl (–SH) groups. These unbound groups have a tendency to interact with other unbound –SH groups that will lead to aggregate formation. In short, your proteins form lumps and become inactive.
The DTT will interact with the free –SH groups of your protein and thus prevent it from interacting with other proteins. Conclusion: DTT will stabilize your protein and keep it in an active conformation.
The problem with DTT is that it needs a pH of 7 or higher to be functional and it tends to oxidize in the presence of air (so keep it in sealed bottles in the refrigerator). Alternatives to DTT include β-mercaptoethanol; however, it has only half the reducing power of DTT, and smells terrible . A better alternative would be TCEP (tris(2-carboxyl)phosphine), which doesn’t have an odor, is as powerful as DTT and will remain functional even at a pH of below 7.
EDTA (ethylenediaminetetraacetic acid)
EDTA is a so-called chelating agent. If you have paid attention in chemistry class, you will know that chelates have a special way of binding with metal ions (if you haven’t paid attention you just have to trust me on this one). Now, why are metal ions important to a biologist? Because many enzymes such as DNases and RNases need metal ions (e.g. magnesium) in order to function. If you add EDTA to your reaction mix, you deplete it of Mg2+ and thereby prevent all those nasty –ases from nibbling away on your nucleic acids.
But as nice as EDTA is, it has its downsides. For Mg2+ is not only needed by mean DNases and RNases, but also by friendly enzymes such as ligases, polymerases or endonucleases. So if you use EDTA in a DNA extraction, you might get problems with downstream reactions, such as ligations, PCR or restriction digests.
Unlike other substances named here, glycogen didn’t make its way into the lab because of its chemical reactivity, but because of its lack of reactivity (it is inert). Actually, it is not a chemical either, but a polysaccharide (a form of sugar), which is naturally produced by many animals (humans included) and fungi. In our body glycogen is stored in the liver and muscles and it can be transformed into glucose to provide energy. So if it’s inert what is it doing in the lab?
Glycogen is often added to ethanol precipitations in order to increase the rate of recovery. As it is not chemically active, it will not inhibit any downstream reactions. It will, however, trap the nucleic acids and form a precipitate with them. This will give you a more visible pellet, and it will help you recover even small amounts of DNA or RNA.
While glycogen is not chemically active, it could possibly interfere with protein–nucleic acid interactions. In which case you might want to switch to another inert carrier (as they are called). Instead of glycogen you might use yeast tRNA or sonicated DNA, which have the advantage of being inexpensive, but they are biologically active so might potentially cause problems during downstream reactions. More expensive but less likely to interfere later is linear (poly)acrylamide.
PMSF (phenylmethylsulfonyl fluoride)
In protocols for protein extractions you will often find PMSF. It inhibits serine proteases by specifically binding to the active-site serine residue of proteases such as trypsin, chymotrypsin and thrombin, thereby inhibiting their functionality. It will also block acetylcholinesterase and thiol proteases such as papain.
PMSF is highly unstable in water and stock solutions should therefore be prepared using anhydrous liquids, such as ethanol, isopropanol or DMSO. It is considered to be highly toxic (imagine what would happened if PMSF inhibited proteases in your body!) so take care while handling it.
Detergents are generally used to solubilize hydrophobic proteins or break-up lipid bilayers such as cell membranes. Ionic detergents have a net charge on their head group and are very effective, but they will denature all proteins they come in contact with.
Non-ionic detergents have no net charge on their head group and are milder. Therefore, they are the preferred choice in protein extractions, as they will break up cell membranes but not denature the proteins extracted.
A famous ionic detergent is SDS (sodium dodecyl sulfate). It can lyse cells, but most often it is used in electrophoresis. It will denature proteins and completely envelope the protein. The negative charge of SDS is much stronger than any potential charge of the protein, which is why all proteins will migrate toward the positive pole during electrophoresis.
Examples of non-ionic detergents include NP40, Triton X-100 or Brij 35. They can all be used to lyse cells without denaturing the proteins in the cell. Another widely used non-ionic detergent is Tween 20 (Polysorbate 20). It is often used in western blots, where it is added to prevent non-specific binding of the antibody.
Evidently this list is far from exhaustive, but these are the reagents that I come across a lot. Maybe you would like to add some compounds that frequently show up in your reactions – if so leave them in the comments.
Leave a Comment
You must be logged in to post a comment.
Thanks so much, this was very simple, clear and easy to understand!! Now I know what I am doing next time I use these reagents!
Nice article. The section on reducing agents is a little bit incomplete though. It is worth noting that reducing agents such as DTT and TCEP can also react with disulfide bonds in proteins, not just free thiols. An additional comment is that while DTT is a dithiol and BME has a single thiol, that doesn’t exactly mean that DTT is twice as effective as BME. DTT is a bit more than twice as effective since in reducing a cysteine disulfide bond, one DTT thiol forms a disulfide with a cysteine thiol, then there is an intramolecular reaction to return the free cystein thiol (see the DTT wikipedia article). For BME, the second step of the reaction is intermolecular rather than intramolecular, so it is less kinetically favorable, which is why I would argue that DTT is a little more than twice as effective as BME.
Because many reaction reagents have the same color, that make the distinguish of them difficult!
Quite useful, thanks.