| Size | Price | Stock | Qty |
|---|---|---|---|
| 5mg |
|
||
| 10mg |
|
||
| 100mg | |||
| Other Sizes |
| Targets |
Ac-IEPD-AFC is a fluorescent substrate, with its target being granzyme B[1]
|
|---|---|
| ln Vitro |
In the granzyme B assay buffer, human granzyme B's Km for Ac-IEPD-AFC was 585 μM [1].
1. It serves as a substrate for human granzyme B, used to detect the inhibitory activity of SRP-2 against human granzyme B. The enzyme activity is reflected by measuring the fluorescence intensity produced by substrate hydrolysis, with excitation/emission spectra of 390 nm/510 nm[1] 2. In the interaction experiment between SRP-2 and human granzyme B, Ac-IEPD-AFC at a final concentration of 10 μM is used to determine binding stoichiometry, 50 μM for enzyme kinetic analysis under pseudo-first order reaction conditions, and 100 μM for the calculation of second-order rate constants[1] 3. It is used to standardize the concentration of human granzyme B. Using Ac-IEPD-pNA or Ac-IEPD-AFC as substrates, the active concentration of granzyme B is determined by active site titration based on a 1:1 stoichiometry[1] |
| Enzyme Assay |
1. Enzyme Inhibition Activity Assay: In granzyme B assay buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 0.01% Tween 20), mix the enzyme (e.g., human granzyme B) with the inhibitor (e.g., SRP-2), incubate at 25℃ for 15 minutes, then add Ac-IEPD-AFC to the specified final concentration. Continuously detect the change in fluorescence intensity with a microplate reader (excitation 390 nm/emission 510 nm), calculate the residual enzyme activity based on the hydrolysis rate. Inhibition rate = 100×(1 - reaction rate of inhibited enzyme / reaction rate of uninhibited enzyme)[1]
2. Binding Stoichiometry Assay: In a 96-well plate, incubate 25 nM human granzyme B with 0-50 nM SRP-2 in granzyme B assay buffer at 25℃ for 15 minutes, add Ac-IEPD-AFC to a final concentration of 10 μM, and detect the substrate hydrolysis rate. Plot fractional activity (reaction rate of inhibited enzyme / reaction rate of uninhibited enzyme) against the initial ratio of inhibitor to enzyme ([I]₀/[E]₀), and determine the stoichiometry of inhibition (SI) by linear regression analysis[1] 3. Enzyme Kinetic Analysis: Under pseudo-first order reaction conditions, fix the concentration of human granzyme B at 25 nM, add 0-800 nM SRP-2 and Ac-IEPD-AFC at a final concentration of 100 μM, continuously detect the change in fluorescence intensity. Calculate the first-order rate constant (kₒᵦₛ) by non-linear regression fitting of the data, plot kₒᵦₛ against the inhibitor concentration, and the slope is the second-order rate constant (k'). The corrected second-order rate constant (kₐ) is obtained by combining the Km value of the substrate (Km of human granzyme B for Ac-IEPD-AFC is 585 μM)[1] |
| References |
[1]. Pak SC, et al. SRP-2 is a cross-class inhibitor that participates in postembryonic development of the nematode Caenorhabditis elegans: initial characterization of the clade L serpins. J Biol Chem. 2004 Apr 9;279(15):15448-59.
|
| Additional Infomation |
1. Ac-IEPD-AFC, short for Acetyl-Ile-Glu-Pro-Asp-7-amino-4-trifluoromethylcoumarin, is a commonly used fluorescent substrate for detecting the activity of apoptosis-related serine proteases (such as granzyme B) [1]. 2. After being hydrolyzed by the target enzyme, this substrate releases the fluorescent group AFC. Changes in fluorescence intensity can quantitatively reflect the enzyme activity level, and therefore it is widely used for screening and characterizing protease inhibitors [1].
|
| Molecular Formula |
C32H38F3N5O11
|
|---|---|
| Molecular Weight |
725.666239261627
|
| Exact Mass |
725.252
|
| CAS # |
1135417-31-0
|
| PubChem CID |
44135102
|
| Appearance |
White to off-white solid powder
|
| Density |
1.4±0.1 g/cm3
|
| Boiling Point |
1073.9±65.0 °C at 760 mmHg
|
| Flash Point |
603.3±34.3 °C
|
| Vapour Pressure |
0.0±0.3 mmHg at 25°C
|
| Index of Refraction |
1.575
|
| LogP |
2.49
|
| Hydrogen Bond Donor Count |
6
|
| Hydrogen Bond Acceptor Count |
14
|
| Rotatable Bond Count |
15
|
| Heavy Atom Count |
51
|
| Complexity |
1420
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
FC(C1=CC(=O)OC2C1=CC=C(C=2)NC([C@H](CC(=O)O)NC([C@H]1CCCN1C([C@H](CCC(=O)O)NC([C@H]([C@H](C)CC)NC(C)=O)=O)=O)=O)=O)(F)F
|
| InChi Key |
HWXKKVLJPWMVLL-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C32H38F3N5O11/c1-4-15(2)27(36-16(3)41)30(49)38-20(9-10-24(42)43)31(50)40-11-5-6-22(40)29(48)39-21(14-25(44)45)28(47)37-17-7-8-18-19(32(33,34)35)13-26(46)51-23(18)12-17/h7-8,12-13,15,20-22,27H,4-6,9-11,14H2,1-3H3,(H,36,41)(H,37,47)(H,38,49)(H,39,48)(H,42,43)(H,44,45)
|
| Chemical Name |
4-[(2-acetamido-3-methylpentanoyl)amino]-5-[2-[[3-carboxy-1-oxo-1-[[2-oxo-4-(trifluoromethyl)chromen-7-yl]amino]propan-2-yl]carbamoyl]pyrrolidin-1-yl]-5-oxopentanoic acid
|
| 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 |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
|
|---|---|
| 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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.3780 mL | 6.8902 mL | 13.7804 mL | |
| 5 mM | 0.2756 mL | 1.3780 mL | 2.7561 mL | |
| 10 mM | 0.1378 mL | 0.6890 mL | 1.3780 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
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.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
