Freeze-thaw comes up often in conversation and rarely with the context attached. Here we lay out the basics in order, then work through the practical considerations.
Last reviewed on 2025-10-13. Where a claim depends on a specific study, the study is described rather than over-claimed.
NAD+ is a dinucleotide composed of two nucleotides joined by a pyrophosphate linkage. One nucleotide contains adenine, and the other contains nicotinamide. The oxidized form carries a positive charge on the nicotinamide ring and is abbreviated NAD+. It functions as a cofactor in hydride-transfer reactions, accepting electrons in catabolic pathways. In cells, it interconverts with reduced NADH, forming a redox couple central to energy metabolism. The molecule is water-soluble and does not cross cell membranes freely without specific transport or precursor pathways.
The nicotinamide ring undergoes reversible reduction at the para position, converting NAD+ to NADH. This reaction transfers a hydride equivalent, not a free hydrogen atom or electron alone. Because the redox pair has a defined reduction potential, it links oxidation of fuels to respiratory chain activity. Many dehydrogenases use NAD+ as a co-substrate and produce NADH. The ratio of NAD+ to NADH reflects metabolic state and influences flux through several pathways.
NAD+ also serves as a substrate for enzymes that cleave it, including sirtuins, PARPs, and CD38. These enzymes consume NAD+ and release nicotinamide and ADP-ribose or related products. The dual roles as redox cofactor and signaling substrate connect NAD+ to DNA repair, circadian regulation, and calcium signaling. Cellular NAD+ concentrations vary by tissue, time of day, and stress exposure. How these consumption pathways interact with redox balance remains an active area of research.
In cells, NAD+ functions primarily as an electron carrier. Dehydrogenase enzymes in glycolysis and the citric acid cycle transfer hydride from substrates to NAD+, producing NADH. NADH then delivers electrons to the mitochondrial respiratory chain, supporting ATP synthesis. In fermentation, NADH is reoxidized to NAD+ so that glycolysis can continue. The balance between NAD+ and NADH helps set metabolic flux. Beyond redox, NAD+ serves as a substrate for enzymes that cleave it, including sirtuins, poly(ADP-ribose) polymerases, and CD38. These reactions consume NAD+ and release nicotinamide and ADP-ribose products.
Biosynthesis occurs through salvage, Preiss-Handler, and de novo pathways. In mammals, the salvage pathway from nicotinamide predominates, and NAMPT is often described as rate-limiting. Nicotinamide riboside and nicotinic acid enter related routes that converge on NAD+ production. Tissue NAD+ concentrations vary widely and are maintained by a balance of synthesis and consumption. Some studies report age-related declines in certain tissues, but whether these changes cause disease or can be reversed to improve human health remains an open question.
| Property | Value | Notes |
|---|---|---|
| IUPAC name | Nicotinamide adenine dinucleotide | Oxidized dinucleotide form |
| CAS Registry Number | 53-84-9 | Common entry for beta-NAD+ |
| Molecular formula | C21H27N7O14P2 | Free acid form |
| Molar mass | 663.43 g/mol | Calculated for free acid |
| Water solubility | Freely soluble | Charged dinucleotide; less soluble in organic solvents |
NAD+ is commonly measured by high-performance liquid chromatography with ultraviolet detection, often at 254 or 260 nm. Enzymatic cycling assays provide higher sensitivity by coupling NAD+ to a reporter reaction. Mass spectrometry can distinguish NAD+ from close analogues and confirm isotope labeling. Sample preparation usually involves rapid quenching of metabolism to prevent interconversion with NADH. Because NAD+ and NADH differ by one hydride, extraction conditions strongly affect the measured ratio.
In aqueous solution, NAD+ is most stable under mildly acidic to neutral conditions and degrades faster at high pH or elevated temperature. The molecule can hydrolyze at the pyrophosphate bond or undergo nonenzymatic cyclization. Buffers, chelating agents, and cold temperatures slow these losses during analysis. Repeated freeze-thaw cycles are generally avoided because they can promote degradation and concentration changes. Light exposure is also controlled, though NAD+ is less photolabile than some related nucleotides.
Commercial NAD+ is supplied as a solid, often as the free acid or a salt, and purity is verified by chromatographic methods. Laboratories typically store it desiccated at minus 20 degrees Celsius or below. Working solutions are prepared fresh because even sterile aqueous solutions can lose activity over hours to days depending on pH and temperature. Documentation may include a certificate of analysis, an assay value, and a recommended retest date. Researchers should verify identity and purity when results depend on precise cofactor concentrations.
The molecule was first described in the early twentieth century as a factor that promoted fermentation in yeast extracts. Later work linked it to hydrogen transfer and to the oxidation of nutrients in living tissues. Its structure was resolved as a dinucleotide, which explained why it could accept and donate electrons at specific enzyme sites. Today, NAD+ is recognized as a central substrate and signaling precursor, not merely a metabolic cofactor. Whether all observed NAD+ changes reflect causal signaling remains an open question.
Related compounds include NADH, the reduced form, and NADP+, which carries an additional phosphate group. NADP+ and NADPH often serve in biosynthetic and antioxidant reactions, while NAD+ and NADH are more associated with energy-yielding catabolism. Nicotinamide, nicotinic acid, and nicotinamide riboside are precursors that can enter salvage pathways. The exact contribution of dietary precursors to tissue NAD+ pools is an area of active investigation. Some studies measure labeled precursors to trace those routes.
NAD+ is the oxidized form of nicotinamide adenine dinucleotide, a coenzyme built from two nucleotides joined by a phosphate linkage. One nucleotide carries adenine, and the other carries nicotinamide; the plus sign denotes a formal positive charge on the nicotinamide ring, not a free proton. In cells, NAD+ and its reduced partner NADH form a reversible redox pair. That pair participates in electron transfer reactions throughout metabolism. The abbreviation NAD+ is common in biochemistry, while NAD(H) sometimes denotes the combined pool.
Cells produce NAD+ through several biosynthetic routes. The salvage pathway recycles nicotinamide, while the Preiss-Handler pathway uses nicotinic acid, and a de novo route can start from tryptophan in some organisms. In mammals, the salvage pathway is generally considered the main source under ordinary conditions. Tissue concentrations vary widely by cell type and compartment, and measured declines with age have been reported in some studies. Whether such changes drive aging or mainly accompany it remains an open question.
Nicotinamide adenine dinucleotide, commonly abbreviated NAD+, is a coenzyme present in all living cells. The molecule consists of two nucleotides linked by phosphate groups, with adenine and a nicotinamide ring as its principal features. In its oxidized form, the nicotinamide ring can accept a hydride ion, becoming NADH. This reversible conversion places NAD+ at the center of many electron-transfer reactions. Its role as a redox carrier is well established across bacteria, plants, fungi, and animals.
Beyond redox chemistry, NAD+ acts as a substrate for several enzyme families. ADP-ribosyltransferases, sirtuins, and CD38 ectoenzymes cleave the molecule into nicotinamide and ADP-ribose or related products. These reactions connect NAD+ availability to processes such as DNA repair, chromatin modification, and calcium signaling. Because the coenzyme is used in both electron transfer and signaling, cells maintain separate pools in compartments including the cytosol, mitochondria, and nucleus. The relative sizes and regulation of those pools remain active areas of study.
In 1928 he obtained a Rockefeller Travelling Fellowship to visit Europe, where he worked for a while in the Anatomical School at Cambridge University, England, then headed by Professor Wilson, and in the Anatomical School at Utrecht University, the Netherlands, then headed by Professor Boecke. Also in 1928, Oscar Tiegs was awarded the David Syme Research Prize. As a histologist Oscar Tiegs developed an interest in Boecke's suggestion of a double innervation of vertebrate skeletal muscle. From this interest he undertook a physiological investigation of the sympathetic system in the muscle, finding no such action except in blood vessels, all muscular action seemingly being caused by an adrenaline-like substance. Oscar Tiegs in this work corrected errors and ambiguities in the work of others, and showed the validity of some traditional interpretations.
=== Genetics === In MCTD, major histocompatibility complex (MHC) and non-MHC genes have been linked to disease vulnerability. HLA-DR4 in the MHC is linked to both anti-RNP antibody responses and MCTD. The HLA class II phenotype/genotype most closely connected with scleroderma, HLA-DR5, and its subgroups, has been demonstrated to have a negative connection with MCTD. Another genetic feature of MCTD is the presence of anti-RNP antibodies. However, these antibodies are not present in all patients. Genome-wide association studies have revealed that there are parts of a patient’s genetic material that cause production of these anti-RNP antibodies. The mechanism is not yet thoroughly defined.
volume The quantity of three-dimensional space enclosed by a closed surface, or the space that a substance (solid, liquid, gas, or plasma) or shape occupies or contains. The SI unit for volume is the cubic metre (m3).
=== Multicolour Labelling === Since EosFP can be used in fusion constructs while maintaining functionality of the protein of interest, it is a popular choice for multi-colour labelling studies. In a dual-colour labelling experiment to map the stages of mitosis, HEK293 cells were first stably transfected with tubulin-binding protein cDNA fused to EGFP for visualization of the spindle apparatus. Then, transient transfection of recombination signal-binding protein (RBP) fused to d2EosFP was used to visualize the beginning of mitosis. Photoconversion was completed by fluorescent microscopy and highlighted the separation between two sets of chromosomes during anaphase, telophase and cytokinesis.
Sources: en.wikipedia.org
Fructilactobacillus sanfranciscensis is a heterofermentative species of lactic acid bacteria which, through the production mainly of lactic and acetic acids, helps give sourdough bread its characteristic taste. It is named after San Francisco, where sourdough was found to contain the variety, though it is dominant in Type I sourdoughs globally. In fact, F. sanfranciscensis has been used in sourdough breads for thousands of years, and is used in 3 million tons of sourdough goods yearly. For commercial use, specific strains of F. sanfranciscensis are grown on defined media, freeze-dried, and shipped to bakeries worldwide.
== Selected publications == Moloney, P. J. (1926). "The preparation and testing of diphtheria toxoid (Anatoxine-Ramon)". American Journal of Public Health. 16 (12): 1208–1210. doi:10.2105/AJPH.16.12.1208. PMC 1321494. PMID 18012024. Moloney, P. J.; Fraser, C. J. (1927). "Immunization with diphtheria toxoid (anatoxine Ramon)". American Journal of Public Health. 17 (10): 1027–1030. doi:10.2105/AJPH.17.10.1027. PMC 1321911. PMID 18012293. Taylor, E. M.; Moloney, P. J. (1939). "A New Schick-Toxin". The Journal of Immunology. 37 (3): 223–232. doi:10.4049/jimmunol.37.3.223. S2CID 86572393. Fitzgerald, J. G.; Defries, R. D.; Fraser, D. T.; Moloney, P. J.; McKinnon, N. E. (1932). "Experiences with Diphtheria Toxoid in Canada". American Journal of Public Health and the Nation's Health. 22 (1): 25–28. doi:10.2105/ajph.22.1.25. PMC 1556712. PMID 18013424. Moloney, P. J.; Hennessy, J. N. (1942). "Purification of tetanus toxoid". The Biochemical Journal. 36 (7–9): 544–547. doi:10.1042/bj0360544. PMC 1266838. PMID 16747558. Moloney, P. J.; Hennessy, Joan N. (1944). "Titration of Tetanal Toxins and Toxoids by Flocculation". The Journal of Immunology. 48 (6): 345–354. doi:10.4049/jimmunol.48.6.345. S2CID 88201977. Moloney, P. J.; Coval, M. (1955). "Antigenicity of insulin: Diabetes induced by specific antibodies". The Biochemical Journal. 59 (2): 179–185. doi:10.1042/bj0590179. PMC 1216116. PMID 14351177. Moloney, P. J.; Goldsmith, L. (1957). "On the Antigenicity of Insulin". Canadian Journal of Biochemistry and Physiology. 35 (1): 79–92. doi:10.1139/y57-011. PMID 13396665.
== Mitogen-activated protein kinase 7 == Mitogen-activated protein kinase 7 (MAPK7) is also known as extracellular signal-regulated kinase 5 (ERK5). Sometimes referred to as Big MAPK or BMK due to its large size in comparison to other MAPKs.
Sources: en.wikipedia.org
NAD+ is the oxidized form, while NADH is the reduced form carrying an added hydride. The two form a redox pair that cells use in many energy-yielding reactions.
NAD+ is a small organic cofactor, not a protein or enzyme. It binds temporarily to enzymes such as dehydrogenases to assist electron transfer.
Intact NAD+ is generally not taken up efficiently by most cells because it is charged and water-soluble. Cells often rely on precursors such as nicotinamide or nicotinamide riboside to produce NAD+ internally.
Nicotinamide adenine dinucleotide, with the plus sign indicating the oxidized form. It is a coenzyme present in all living cells. The reduced form is NADH.