Dithiothreitol (DTT) vs Beta-mercaptoethanol (BME)

DTT and BME are reducing agents used for the chemical reduction of disulfide bonds. They are commonly added to SDS-PAGE sample buffers and are often used interchangeably. While both DTT and BME are used to achieve the same purpose in SDS-PAGE, they exhibit different chemical properties.

BME

This is very volatile and readily evaporates from solution. Because of its volatility and toxicity, solutions of BME are often handled in a fume cupboard. The disadvantage of this is that frequent usage will increase the rate of evaporation, leading to a decrease in the concentration of a solution of BME over time.

The issue with this is that the chemical reduction of disulfide bonds within proteins and peptides is an equilibrium reaction where bonds are continually breaking and re-forming. Accordingly, excess BME is required to drive the reaction forward to completion. Reciprocally, insufficient quantities of BME in a given reaction will not adequately reduce all protein disulfide bonds with some bonds undergoing reoxidation.

DTT

This is volatile but not to the extent as BME. Unlike BME, the chemical reaction in reducing disulfide bond linkages within proteins and peptides is not an equilibrium reaction. A disulfide reduction reaction using DTT leads to an irreversible change in the DTT molecule where its straight chain structure is altered to a ring structure. Accordingly, use of DTT will avoid issues of disulfide bond reoxidisation. However, DTT is unstable in solution and must be made fresh each time.

Static On Film

Have you ever gotten strange artifacts on your films that look like this:

This is the result of static electricity and if you have acclimatized yourself for long enough in the darkroom; you can even see when the static electricity sparks go off on the film.

These artifacts are a pain because they can ruin a perfectly good experiment. For something like western blotting, getting an artifact like the above that covers up your result is annoying but most of the time, you can make a few repeat exposures before the ECL substrate gives out on your membrane. However, if you are working with something that takes weeks to months to develop on film (e.g. S³⁵ in situ hybridization), getting an artifact that covers up some of your most important image results can be very devastating as the entire experiment will probably need to be repeated.

I have had the unfortunate experience of having these artifacts crop up in my experiments. Over time, I came to realize that the main problem lied with the lab’s air conditioning.

Static electricity is a build up of electrons (negative charge) within or on the surface of an object that is retained until it can be released by an electric current or electrical discharge. Water is a conductor of electricity, so in a humid environment, the moisture in the air absorbs and can evenly distributes excess charges, thereby allowing excess charge to leave objects.

My Experience

During my time working in a lab housed in an old building, I have never once gotten an artifact like the above on any of my films (and I had done a lot of film work). Fast forward to my new lab, which was brand new, the artifacts appeared almost all the time I did any film work. My technique, equipment, and reagents were pretty much the same so the only real difference was the lab itself. I ultimately concluded that the problems came from the level or moisture in the air. In my old lab, there was no air conditioning or windows that actually opened. As such, the lab was always humid. In contrast, my new lab was fully air conditioned and the air was always very dry. I tested my theory by having the air conditioning turned off in the new darkroom; while it did get pretty hot and humid in there, importantly, the artifacts stopped.

Suggestions

If you are having problems with artifact on your films cause by static electricity:

* You can try using films containing an anti-static layer, although I still found the artifacts cropping up on them.

* Try preventing the static build up beforehand. If you are using transparency film to sandwich your western blotting membranes, leave them in a humid area. The same goes for the autoradiography film or hyperfilms.

* If possible, switch off the air conditioning (at least for the darkroom).

* If possible, use a humidifier.

Western Blotting – Why Are My Gels Not Setting?

This question comes up quite often. It can be frustrating having to work to a tight schedule and when for whatever reason your separating and/or stacking gel decides not to set, plans can be sent into a tailspin. 

Usually when a gel doesn’t set, you just start again, but you will be wondering why it didn’t set in the first place. Some common causes are obvious but some are less so.

Clean Your Plates

Those glass plates really need to be clean. If you have residue left over from the previous western run, it can impact on gel polymerization. The same goes for the combs, they need to be cleaned too.

Just before assembling your plates for pouring, spray and wipe (using lint-free tissues) the plates and combs with 70% ethanol.

APS/TEMED

APS and TEMED are important components of the gel mix as they are required for the polymerization of the acrylamide monomers. APS forms the free radicals needed for the polymerization process while TEMED stabilizes those free radicals to speed up the entire process.

APS goes off very easily. If you have made fresh APS and stored it at 4 degrees, it should be fine for about 1-2 weeks (depending on the frequency of use), provided you have not left it to sit at room temperature for extended periods. APS sitting at room temperature for hours should be thrown out.

Ideally, it is better to make fresh APS for each experiment. Just weigh out what you need for each experiment and reconstitute with water.

If despite using fresh APS you find that your gels are setting very slowly i.e. taking hours instead of minutes, you may want to consider using new TEMED.