2,6-Diaminopimelic acid |
Catalog No.GA20387
One-Click Copy Product Info
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2,6-Diaminopimelic acid is a symmetrical α, α′-diamino dicarboxylic acid found in bacteria and higher plants.
Products are for research use only. Not for human use. We do not sell to patients.
Cas No.: 583-93-7
Sample solution is provided at 25 µL, 10mM.
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2,6-Diaminopimelic acid is a symmetrical α, α′-diamino dicarboxylic acid found in bacteria and higher plants [1]. 2,6-Diaminopimelic acid is a precursor for the biosynthesis of L-lysine and an important component of bacterial peptidoglycan [2]. 2,6-Diaminopimelic acid is a part of the bacteria that changes the bacteria (as a germ of bacteria). Presence or absence of 2,6-Diaminopimelic acid in the cell wall, classification of staphylococcal bacteria (containing 2,6-Diaminopimelic acid) and staphylococcal bacteria (containing staphylococcus acid) [3-4].
References:
[1]. Dzierzbicka K. Synthesis of 2, 6-diaminopimelic acid (DAP) and its analogues. Polish Journal of Chemistry. 2007; 81: 455-473.
[2]. Shaikh MS, Kale MA, Sharuk K. Discovery of Heterocyclic Analogs of Diaminopimelic Acid as Promising Antibacterial Agents Through Enzyme Targeted Inhibition of Lysine Biosynthesis. Current Enzyme Inhibition. 2018 Aug 1; 14(2): 120-130.
[3]. Masson HA, Denholm AM, Ling JR. In vivo metabolism of 2, 2'-diaminopimelic acid from gram-positive and gram-negative bacterial cells by ruminal microorganisms and ruminants and its use as a marker of bacterial biomass. Applied and environmental microbiology. 1991 Jun; 57(6): 1714-1720.
[4]. Bhamidi S, Shi L, Chatterjee D, et al. A bioanalytical method to determine the cell wall composition of Mycobacterium tuberculosis grown in vivo. Analytical biochemistry. 2012 Feb 1; 421(1): 240-249.
This protocol is used to distinguish Staphylococci containing 2,6-Diaminopimelic acid (DAP) from Staphylococci containing teichoic acid [1-2]. It is only used as an experimental guide. The actual application needs to be modified according to your needs.
1. Preparation of reagents and materials
Pure culture of Staphylococcus spp. to be tested; DAP standard solution (1mg/mL, dissolved in 0.1M HCl); teichoic acid standard (or a positive control strain known to contain teichoic acid, such as Staphylococcus aureus); 6M hydrochloric acid (HCl), 10% trichloroacetic acid (TCA); ninhydrin colorimetric solution; phosphomolybdic acid staining solution.
2. Experimental steps
Bacterial cell wall extraction and hydrolysis:
(1) Bacterial collection:
The strain to be tested was inoculated into TSB liquid medium and cultured at 37℃ for 18-24 hours. The bacteria were collected by centrifugation (8000rpm, 10min) and washed 3 times with PBS. Freeze-dried cells or directly proceed to the next step of hydrolysis.
(2) Acid hydrolysis (DAP release):
Take 50mg of wet cells, add 1mL of 6M HCl, and hydrolyze at 110℃ for 16-18 hours. After cooling, centrifuge (12000rpm, 5min), collect the supernatant, and filter with a 0.22μm filter membrane.
(3) TCA treatment (extract staphylococcal acid):
Take another 50mg of wet cells, add 1mL of 10% TCA, and boil at 100℃ for 30min. After centrifugation, take the supernatant for later use.
Thin layer chromatography (TLC) detection:
(1) DAP detection:
Spotting: Spot the acid hydrolyzate and DAP standard on the TLC plate (silica gel G layer).
Developing solvent: n-butanol: glacial acetic acid: water = 4:1:1 (v/v).
Color development: spray ninhydrin solution, bake at 110℃ for 5min, and observe purple spots (Rf value of DAP is about 0.3).
(2) Staphylococcal acid detection:
Spotting: TCA extract and positive control are spotted on another TLC plate.
Developing solvent: chloroform: methanol: water = 65:25:4 (v/v).
Color development: spray phosphomolybdic acid staining solution, bake at 105℃ for 10min, blue spots indicate the presence of phosphate groups.
Result judgment:
(1) Staphylococci containing DAP: After TLC color development, the acid hydrolysis sample is consistent with the DAP standard spot position (ninhydrin shows purple), and the staphylococcal acid test is negative.
(2) Staphylococci containing staphylococcal acid: TCA extract shows blue spots (phosphomolybdic acid staining is positive), and the DAP test is negative.
Precautions:
(1) The acid hydrolysis of DAP requires strict temperature control (110℃±2℃) to avoid excessive degradation. When extracting staphylococcal acid, TCA treatment for too long may destroy the phosphate group.
(2) It is recommended to combine Gram staining (to confirm bacterial morphology) and biochemical identification (such as catalase test) to assist in differentiation.
(3) If the TLC signal is weak, HPLC or mass spectrometry (LC-MS) can be used for quantitative analysis.
(4) Hydrochloric acid and trichloroacetic acid are corrosive, so gloves and goggles must be worn during operation.
References:
[1]. Masson HA, Denholm AM, Ling JR. In vivo metabolism of 2, 2'-diaminopimelic acid from gram-positive and gram-negative bacterial cells by ruminal microorganisms and ruminants and its use as a marker of bacterial biomass. Applied and environmental microbiology. 1991 Jun; 57(6): 1714-1720.
[2]. Bhamidi S, Shi L, Chatterjee D, et al. A bioanalytical method to determine the cell wall composition of Mycobacterium tuberculosis grown in vivo. Analytical biochemistry. 2012 Feb 1; 421(1): 240-249.
| Cas No. | 583-93-7 | SDF | |
| Formula | C7H14N2O4 | M.Wt | 190.2 |
| Solubility | H2O : 10.42 mg/mL (54.78 mM; ultrasonic and warming and heat to 60°C) | Storage | Store at -20°C |
| General tips | Please select the appropriate solvent to prepare the stock solution according to the
solubility of the product in different solvents; once the solution is prepared, please store it in
separate packages to avoid product failure caused by repeated freezing and thawing.Storage method
and period of the stock solution: When stored at -80°C, please use it within 6 months; when stored
at -20°C, please use it within 1 month. To increase solubility, heat the tube to 37°C and then oscillate in an ultrasonic bath for some time. |
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| Shipping Condition | Evaluation sample solution: shipped with blue ice. All other sizes available: with RT, or with Blue Ice upon request. | ||
| Prepare stock solution | |||
|
1 mg | 5 mg | 10 mg |
| 1 mM | 5.2576 mL | 26.2881 mL | 52.5762 mL |
| 5 mM | 1.0515 mL | 5.2576 mL | 10.5152 mL |
| 10 mM | 525.8 μL | 2.6288 mL | 5.2576 mL |
Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)
g
μL
Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)
Calculation results:
Working concentration: mg/ml;
Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid 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 master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL saline, mix and clarify.
Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.
Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such as vortex, ultrasound or hot water bath can be used to aid dissolving.
3. All of the above co-solvents are available for purchase on the GlpBio website.
Quality Control & SDS
- View current batch:
- Purity: >98.00% Appearance: A solid
- COA (Certificate of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
Average Rating: 5 (Based on Reviews and 18 reference(s) in Google Scholar.)