Branched Chain Amino Acid (Leu/Ile/Val) Assay Kit

Antigen: Branched Chain Amino Acid (Leu/Ile/Val)

Applikation: Biochemisches Assay (BCA)

Produktnummer: ABIN411641

Produktbeschreibung

Beschreibung: The branched-chain amino acids or BCAA’s, refer to the amino acids with non-linear aliphatic side-chains, namely leucine, isoleucine and valine. These three essential amino acids make up approximately 1/3 of skeletal muscle in the human body. BCAA’s are currently used clinically to aid in the recovery of burn victims, as well as for strength supplementation for athletes. BCAA’s, primarily Leu, can stimulate insulin secretion. The BCAA’s have also been implicated in a wide range of other physiological effects. BioVision's BCAA Assay Kit provides a simple convenient means of measuring the BCAA’s in a variety of biological samples. The kit utilizes an enzyme assay in which BCAA is oxidatively deaminated, producing NADH which reduces the probe, generating a colored product (λ max = 450 nm). BioVision’s BCAA kit measures BCAA’s in the range of 0 to 10 nmol per sample with a detection limit of ~0.2 nmol (~10 µM BCAA in sample). BCAA’s are present in serum ~ 0.1-0.4 mM each (~0.125-1.5 mM combined).

Anwendungen

Protokoll: 1. Standard Curve: Dilute 10 μl of the 10 mM Leucine Standard with 90 μl dH2O to generate 1 mM Leucine standard. Add 0, 2, 4, 6, 8, 10 μl of the diluted Standard into a 96-well plate to generate 0, 2, 4, 6, 8, 10 nmol/well standard. Bring the volume to 50 μl with Assay Buffer. 2. Sample Preparation: Tissue (20 mg) or cells (2 x 10 6 ) can be homogenized with 100 µl Assay buffer. Centrifuge at 15,000g for 10 minutes to remove cell debris and other insoluble materials. Add samples to sample wells in a 96-well plate and bring the volume to 50 μl/well with Assay Buffer. We suggest testing several doses of your sample to make sure the readings are within the standard curve range. Typical volume for serum samples should be in the range of 1 – 20 µl. 3. Reaction Mix: Mix enough reagents for the number of assays to be performed. For each well, prepare a total 50 μl Reaction Mix containing: Amino Acid Measurement Bkgd Control Assay Buffer 46 μl 48 μl Enzyme Mix 2 μl ----- WST Substrate Mix 2 μl 2 μl Add 50 μl of the Reaction Mix to each well containing the leucine standard and test samples. Mix well. Incubate the reaction for 30 min at room temperature, protect from light. NADH and NADPH can generate significant background. If these compounds are suspected of being in your sample at significant concentration, perform a simple background control by replacing the Enzyme Mix with 2 µl Assay Buffer. The background reading should be subtracted from the BCAA test sample readings. 4. Measure O.D. at 450 nm in a microplate reader 5. Calculation: Correct background by subtracting the value derived from the 0 BCAA standard from all readings (The background reading can be significant and must be subtracted from sample readings). Plot standard curve. Apply sample readings to the standard curve. BCAA concentrations of the test samples can then be calculated: C = S a /S v (nmol/μl, or mM) Where: S a = BCAA content of unknown samples (nmol) from standard curve, S v = sample volume (μl) added into the assay wells. BCAA molecular weights are: Leu 131.18, Ile 131.18, Val 117.15 g/mol. Leucine Assay performed according to this protocol

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