H-Gln-βNA . HCl |
Catalog No.GA22342
One-Click Copy Product Info
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H-Gln-βNA . HCl is a specific fluorescent substrate for glutaminyl cyclase, with the excitation wavelength of 320nm and emission wavelength of 410nm.
Products are for research use only. Not for human use. We do not sell to patients.
Cas No.: 201988-95-6
Sample solution is provided at 25 µL, 10mM.
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H-Gln-βNA . HCl is a specific fluorescent substrate for glutaminyl cyclase, with the excitation wavelength of 320nm and emission wavelength of 410nm[1]. H-Gln-βNA . HCl can be used to detect the activity-dependent secretion of glutaminyl cyclase in the secretory products of chromaffin cells[2]. H-Gln-βNA . HCl can be used to evaluate the glutaminyl cyclase inhibitors related to metal chelating agents and imidazole derivatives[3].
References:
[1] Koch B, Kolenko P, Buchholz M, et al. Crystal structures of glutaminyl cyclases (QCs) from Drosophila melanogaster reveal active site conservation between insect and mammalian QCs[J]. Biochemistry, 2012, 51(37): 7383-7392.
[2] Cynis H, Funkelstein L, Toneff T, et al. Pyroglutamate-amyloid-β and glutaminyl cyclase are colocalized with amyloid-β in secretory vesicles and undergo activity-dependent, regulated secretion[J]. Neurodegenerative Diseases, 2014, 14(2): 85-97.
[3] Huang K F, Hsu H L, Karim S, et al. Structural and functional analyses of a glutaminyl cyclase from Ixodes scapularis reveal metal-independent catalysis and inhibitor binding[J]. Biological Crystallography, 2014, 70(3): 789-801.
Determination of Glutaminyl cyclase activity using H-Gln-βNA . HCl as the substrate[1]
This protocol only provides a guideline, and should be modified according to your specific needs.
1. Prepare solution
(1) Prepare stock solution: Prepare H-Gln-βNA . HCl stock solution with a concentration of 1mM in high-quality anhydrous DMSO. Notice: Aliquot the unused stock solution and store it in the dark at -20°C to avoid repeated freezing and thawing.
(2) Prepare working solution: Dilute the stock solution with PBS to prepare a working solution with a concentration of 100μM. Please adjust the concentration of the working fluid according to the actual situation, and prepare it now to avoid repeated freezing and thawing. Notice: The working solution must be prepared fresh on the day of the experiment.
2. Sample pretreatment
Test samples from brain or peripheral tissue were homogenized in a buffer consisting of 10mM Tris, 100mM NaCl, 5mM EDTA, 0.5% Triton X-100, and 10% glycerol, pH 7.5, using a homogenizer. The homogenate was further sonicated and centrifuged at 16000×g for 30min and 4°C. The protein concentration of the resulting supernatant containing glutaminyl cyclase and iso-glutaminyl cyclase was adjusted to 5-7mg/ml.
3. Reaction and Detection
Reaction samples consisted of 50μM H-Gln-βNA . HCl in 25mM MOPS, pH 7.0, 0.1mM N-ethylmaleinimide, and enzyme solution in a final volume of 1ml. The reaction temperature was 37°C. Test samples were removed for up to 1h, and the reaction was stopped by boiling for 5min followed by centrifugation at 16000×g for 10min. The supernatant was applied to HPLC analyses. The samples (20μl) were injected and separated by increasing the concentration of solvent A (acetonitrile containing 0.1% TFA) from 8 to 20% in solvent B (H2O containing 0.1% TFA). Glutaminyl cyclase activity was quantified from a standard curve of pGlu-βNA determined.
4. Precautions:
(1) Please avoid light as much as possible to slow down fluorescence quenching.
(2) For your safety and health, please wear a lab coat and disposable gloves when operating
(3) H-Gln-βNA . HCl should be stored in the dark throughout the experiment.
(4) This protocol and all reagents are for scientific research use only.
References:
[1] Schilling S, Kohlmann S, Bäuscher C, et al. Glutaminyl cyclase knock-out mice exhibit slight hypothyroidism but no hypogonadism: implications for enzyme function and drug development[J]. Journal of Biological Chemistry, 2011, 286(16): 14199-14208.
| Cas No. | 201988-95-6 | SDF | |
| Formula | C15H17N3O2 · HCl | M.Wt | 307.8 |
| Solubility | Soluble in DMSO | Storage | Store at -20°C,protect from light |
| 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 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 3.2489 mL | 16.2443 mL | 32.4886 mL |
| 5 mM | 649.8 μL | 3.2489 mL | 6.4977 mL |
| 10 mM | 324.9 μL | 1.6244 mL | 3.2489 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 35 reference(s) in Google Scholar.)