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.

miRNA/siRNA Knockdown – When Protein Expression Differs From mRNA Levels

A perfect knockdown consists of mRNA reduction and a corresponding change in protein expression. Unfortunately, this is not always the case and there are often times when proteins show an increase or no change. This does not necessarily mean that a knockdown was not effective; rather, it may have occurred because of other cellular processes relating to translation or protein half-life.

Here are a few reasons why your mRNA and protein expression measures may differ:

* If there are multiple alternative transcripts and isoforms of a protein, selective knockdown of one transcript/isoform may lead to increased translation of the other transcripts. If all your isoforms are approximately the same size on a western blot and you do not have a specific antibody targeting your isoform of interest, don’t be surprised if you see an increase in protein expression. The same applies to transcript levels if you are using pan primers.

* MicroRNAs can regulate gene expression by inhibiting translation of existing mRNA and/or promote mRNA degradation. Hence, decreases in target protein expression may not necessarily match the direction of change of mRNA levels. Which route is taken may depend on the degree of base-pairing between the miRNA and miRNA-binding site.

* There can be more than one miRNA recognition element (MRE) for the same or different miRNAs within the 3’UTR of a transcript of interest. Different MREs can function cooperatively to enhance repression. Accordingly, more than one miRNA may be needed for effective knockdown.

* Protein-half life is also relevant. For instance, a target protein containing a PEST sequence will have a short intracellular half-life compared to those that do not.

* The type of protein or function of the protein may also be important. For instance, cell signaling proteins typically have a short-half life whereas glycolytic proteins typically have a long half life.

* Culturing conditions may also influence mRNA and protein expression. For instance, oxidative stress, growth factors, etc will have an impact.

References:

1. Valencia-Sanchez et al. (2006) Control of translation and mRNA degradation by miRNAs and siRNAs. Genes and Development 20:515-524. 

2. To et al. (2008) Regulation of ABCG2 expression at the 3′ untranslated region of its mRNA through modulation of transcript stability and protein translationby a putative microRNA in the S1 colon cancer cell-line. Molecular and Cellular Biology 28:5147-5161. 

3. Wong J. (2014) Regulation of a TrkB Alternative Transcript by microRNAs. Dementia and Geriatric Cognitive Disorders Extra 4:364-74. 

4. Lal et al. (2009) miR-24 inhibits cell proliferation by targeting E2F2, MYC, and other cell-cycle genes via binding to ‘seedless' 3′UTR microRNA recognition elements. Molecular Cell 35:610-625. 

5. Franch et al. (2001) A mechanism regulating proteolysis of specific proteins during renal tubular cell growth. The Journal of Biological Chemistry 276, 19126-19131. 

6. Ito et al. (1996) Oxidative stress increases glyceraldehyde-3-phosphate dehydrogenase mRNA levels in isolated rabbit aorta.  American Journal of Physiology 270(1 Pt2):H81-7.