| Size | Price | |
|---|---|---|
| 500mg | ||
| 1g | ||
| Other Sizes |
| ln Vitro |
Application:
1. ALDH can be used to detect acetaldehyde in blood for the purpose of disease diagnosis. 2. Detoxification of alcohol/hangovers (alcohol detox): Aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase can be combined to compensate for the body's lack of certain enzymes and mitigate the negative effects of alcohol to the body. |
|---|---|
| Enzyme Assay |
Thermofluor assay[1]
A thermofluor assay was used to assess the relative thermostability of the ALDHTt samples. The dye Sypro orange and a qPCR machine with an excitation filter of 498 nm and an emission filter of 610 nm were used. Samples were equilibrated at 25 °C for 3 min before a ramped heating step between 25–95 °C. The heating rate was set to 0.03 °C/s corresponding to 20 acquisitions/°C. The first derivative and Tm were plotted using OriginPro 9.1. Enzyme kinetics[1] Enzyme assays were performed over a short period of time to ensure no evaporative loss of the volatile aldehydes. The standard assay condition was chosen and optimized from the conditions which gave the largest and most stable change in the fluorescence readings. The standard enzyme assay consisted of 10 mM KH2PO4-K2HPO4 (Kpi) pH 8.0, 0.4 mM NAD+, 60 nM ALDH, 1 mM hexanal, in a total volume of 0.5 ml unless otherwise stated. A substrate-lacking reaction was used as a negative control. The reaction was monitored at 50 °C for 60 s. The reduction of NAD+ cofactor to NADH was monitored in a Cary fluorescence spectrophotometer (λexcitation = 340 nm; λemission = 463 nm) with respective slit widths of 20 nm and 5 nm. All components were heated before being added to the cuvette and the reaction was started with the addition of substrate. One unit of enzyme was defined as the amount of enzyme which catalyzed the formation of 1.0 µmol of NADH/min. All assays were performed as a minimum of two independent experiments with triplicates for each reaction. Kinetics data and curve fitting were performed using OriginPro 9.1. |
| Cell Assay |
loning, production and crystallisation of wild type recombinant ALDHTt and its truncated mutants[1]
Primers used for amplification and cloning of the gene encoding for ALDHTt (TTC0513; Accession ID: WP_011172958) into the pET-22b (+) vector are summarized in Supplementary Table S2. The construct DNA for each of the wild type and truncated mutants of ALDHTt were transformed into Escherichia coli BL21 Star (DE3) competent cells. The auto-induction ZYM-5052 medium51, supplemented with ampicillin (100 µg/ml), was inoculated with an overnight culture of the transformed cells at 1% (v/v), and grown for 48 h at 25 °C with shaking at 200 rpm. Cells were collected by centrifugation (6500 × g, 15 minutes, 4 °C) and washed in 500 ml of 20 mM Tris-HCl pH 7.5, 5 mM β-mercaptoethanol, 10 mM imidazole and 500 mM NaCl. Following a second centrifugation step, the cells were resuspended in the same buffer supplemented with Lysozyme (1 mg/ml) and 5 mM EDTA pH 8.0 and stirred at room temperature for 1 h. DNase (0.1 mg/ml) was then added along with 5 mM MgCl2 and the cells were gently stirred at 4 °C for 1 h and sonicated in an ice-water bath for 20 min. Finally, the mixture was heated to 65 °C for 15 min and centrifuged at 25,000 × g, 30 min, 4 °C). The supernatant was then loaded onto an XK 16/20 column containing Ni Sepharose 6 Fast Flow pre-equilibrated with 20 mM Tris-HCl pH 7.5, 5 mM β-mercaptoethanol, 10 mM imidazole and 200 mM NaCl. Bound proteins were eluted using a step gradient of 60, 100, 250 and 500 mM of imidazole in 20 mM Tris-HCl, 5 mM β-mercaptoethanol and 150 mM NaCl, pH 7.5. Fractions containing the protein were then dialyzed overnight in 50 mM Tris-HCl, 5 mM β-mercaptoethanol and 250 mM NaCl, pH 7.5 prior to concentration using Amicon Ultra-15 centrifugal filters, 50 kDa MWCO and loaded onto a HiLoad 16/60 Superdex 200 pg column pre-equilibrated with 50 mM Tris-HCl pH 7.5, 5 mM β-mercaptoethanol and 150 mM NaCl. |
| References | |
| Additional Infomation |
Aldehyde dehydrogenases (ALDH) form a superfamily of dimeric or tetrameric enzymes that catalyze the oxidation of a broad range of aldehydes into their corresponding carboxylic acids with the concomitant reduction of the cofactor NAD(P) into NAD(P)H. Despite their varied polypeptide chain length and oligomerisation states, ALDHs possess a conserved architecture of three domains: the catalytic domain, NAD(P)+ binding domain, and the oligomerization domain. Here, we describe the structure and function of the ALDH from Thermus thermophilus (ALDHTt) which exhibits non-canonical features of both dimeric and tetrameric ALDH and a previously uncharacterized C-terminal arm extension forming novel interactions with the N-terminus in the quaternary structure. This unusual tail also interacts closely with the substrate entry tunnel in each monomer providing further mechanistic detail for the recent discovery of tail-mediated activity regulation in ALDH. However, due to the novel distal extension of the tail of ALDHTt and stabilizing termini-interactions, the current model of tail-mediated substrate access is not apparent in ALDHTt. The discovery of such a long tail in a deeply and early branching phylum such as Deinococcus-Thermus indicates that ALDHTt may be an ancestral or primordial metabolic model of study. This structure provides invaluable evidence of how metabolic regulation has evolved and provides a link to early enzyme regulatory adaptations.[1]
|
| CAS # |
9028-88-0
|
|---|---|
| Appearance |
White to light yellow solid powder
|
| Synonyms |
Aldehyde dehydrogenase (NAD(P))
|
| HS Tariff Code |
2934.99.9001
|
| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
H2O: ≥ 20 mg/mL
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|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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