Ac-ANW-AMC (Synonyms: Ac-Ala-Asn-Trp-AMC) |
| カタログ番号GC42685 |
Ac-ANW-AMC は、免疫プロテアソームの蛍光基質です。
Products are for research use only. Not for human use. We do not sell to patients.
Cas No.: 2357123-49-8
Sample solution is provided at 25 µL, 10mM.
;)
,-1-7$$$37316672.jpg');)
;)
;)
$$$36806353.jpg');)
;33(7)%EF%BC%9A546-561$$$37156877.jpg');)
;)
;)
;)
%201124-1138.$$$35908550.jpg');)
%20766-780.$$$37100057.jpg');)
%20418-432.$$$36893736.jpg');)
%EF%BC%9A-240-255.$$$35108512.jpg');)
533-545$$$36543525.jpg');)
%201-13.$$$36600053.jpg');)
;)
Ac-ANW-AMC is a fluorescent substrate for the β5i (LMP7/PSMB8) subunit of the 20S immunoproteasome, with a maximum excitation wavelength of λEx=345nm and a maximum emission wavelength of λEm=445nm[1, 2]. When a specific enzyme acts on Ac-ANW-AMC, it releases the fluorophore 7-amido-4-methylcoumarin (AMC). The activity of the enzyme can be quantitatively analyzed by measuring the fluorescence intensity of AMC[3]. The fluorescence signal of Ac-ANW-AMC can be effectively eliminated by the i-proteasome inhibitor ONX-0914[4].
References:
[1] Winter M B, La Greca F, Arastu-Kapur S, et al. Immunoproteasome functions explained by divergence in cleavage specificity and regulation[J]. Elife, 2017, 6: e27364.
[2] Erokhov P A, Kulikov A M, Karpova Y D, et al. Proteasomes in patient rectal cancer and different intestine locations: where does proteasome pool change?[J]. Cancers, 2021, 13(5): 1108.
[3] Mustafa M S, El-Abadelah M M, Mubarak M S, et al. Synthesis and fluorogenic properties of some 1-(coumarin-7-yl)-4, 5-dihydro-1, 2, 4-triazin-6 (1H)-ones[J]. International Journal of Chemistry, 2011, 3(4): 89.
[4] Kim S, Park S H, Choi W H, et al. Evaluation of Immunoproteasome-Specific Proteolytic Activity Using Fluorogenic Peptide Substrates[J]. Immune Network, 2022, 22(3).
This plan only provides a guide, please modify it to meet your specific needs.
1. Solution preparation
(1) Working solution: Dissolve Ac-ANW-AMC in DMSO and dilute to the required working concentration with assay buffer, e.g. 30-100μM[1, 2].
Note: Please adjust the optimal working concentration according to the actual situation or refer to the literature to set the gradient concentration by yourself. The working solution must be prepared and used immediately.
2. Measure the catalytic activity of β5i by monitoring the hydrolysis rate of Ac-ANW-AMC over time[1] (from the literature, for reference only)
(1) Prepare protein lysate using Passive Lysis Buffer and dilute in 20S proteasome assay buffer (20mM Tris-HCl, 0.5mM EDTA, 0.035% SDS, pH 8.0).
(2) Add 100μM Ac-ANW-AMC to initiate the catalytic reaction of β5i.
(3) The fluorescence signal was measured using a microplate reader at an excitation wavelength of 360nm and an emission wavelength of 460nm, with readings taken once per minute for 1h.
3. Determination of proteasomal LMP7 activity using Ac-ANW-AMC[2] (from the literature, for reference only)
(1) The reaction mixture contained 20mM Tris-HCl (pH 7.5), 30μM Ac-ANW-AMC, 1mM dithiothreitol (DTT), 5mM MgCl2, and 1mM ATP.
(2) To calculate the contribution of non-proteasomal proteolytic activity, 7μM MG 132 (a proteasome inhibitor) was used. After adding 0.5, 1.0, 1.5, and 2.0μL of the clarified homogenate to a total volume of 100μL, the reaction was incubated at 37°C for 20min and terminated with 1% SDS.
(3) The product was detected using a fluorometer with an excitation wavelength of 380nm and an emission wavelength of 440nm. The difference between the total activity and the residual activity in the presence of MG 132 was determined as the proteasome activity.
References:
[1] Lee M J, Miller Z, Park J E, et al. H727 cells are inherently resistant to the proteasome inhibitor carfilzomib, yet require proteasome activity for cell survival and growth[J]. Scientific reports, 2019, 9(1): 4089.
[2] Erokhov P A, Kulikov A M, Karpova Y D, et al. Proteasomes in patient rectal cancer and different intestine locations: where does proteasome pool change?[J]. Cancers, 2021, 13(5): 1108.
| Cas No. | 2357123-49-8 | SDF | |
| 同義語 | Ac-Ala-Asn-Trp-AMC | ||
| Chemical Name | (S)-N1-((S)-3-(1H-indol-3-yl)-1-((4-methyl-2-oxo-2H-chromen-7-yl)amino)-1-oxopropan-2-yl)-2-((S)-2-acetamidopropanamido)succinamide | ||
| Canonical SMILES | O=C1C=C(C)C2=C(C=C(NC([C@H](CC3=CNC4=C3C=CC=C4)NC([C@H](CC(N)=O)NC([C@H](C)NC(C)=O)=O)=O)=O)C=C2)O1 | ||
| Formula | C30H32N6O7 | M.Wt | 588.6 |
| 溶解度 | 10mM 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. |
||
| 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 | 1.6989 mL | 8.4947 mL | 16.9895 mL |
| 5 mM | 0.3398 mL | 1.6989 mL | 3.3979 mL |
| 10 mM | 0.1699 mL | 0.8495 mL | 1.6989 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 ddH2O, 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: >95.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
Average Rating: 5 (Based on Reviews and 1 reference(s) in Google Scholar.)
GLPBIO products are for RESEARCH USE ONLY. Please make sure your review or question is research based.
Required fields are marked with *