β-Hydroxybutyrate (β-HB) Colorimetric Assay Kit

Details zu Produkt Nr. ABIN411639, Anbieter: Anmelden zum Anzeigen
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Reaktivität
Chemical
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Detektionsbereich
0.01-0.2 mM
Untere Nachweisgrenze
0.01 mM
Applikation
Biochemical Assay (BCA)
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Proben Biological Fluids, Cell Culture Supernatant, Plasma, Serum, Tissue Samples
Nachweismethode Colorimetric
Spezifität Beta-HB Assay kit utilizes beta-HB Dehydrogenase to generate a product which reacts with our colorimetric probe with an absorbance band at 450 nm. The kit is an easy and convenient assay to measure beta-HB levels in biological samples. The assay is linear for 1-20 nmol beta-HB in up to 100 ?L samples or 0.01-0.2 mM of beta-HB samples. beta-Hydroxybutyric Acid.
Produktmerkmale beta-Hydroxybutyrate (beta-HB) Assay Kit: Easy and Convenient Colorimetric Assay to measure beta-HB levels in Biological Samples such as Serum, Urine etc. within 40 min.
Bestandteile β-HB Assay Buffer
β-HB Enzyme Mix
β-HB Substrate Mix
β-HB Standard (1.0 μmol)
Andere Bezeichnung beta-Hydroxybutyrate
Substanzklasse Chemical
Hintergrund Diabetic ketoacidosis occurs when circulating insulin levels drop to very low levels, shutting off the supply of glucose to the body. The physiological response is for the liver to produce ketone bodies (acetoacetate, acetone, and primarily β-hydroxybutyrate) from the acetyl CoA produced from fatty acid oxidation. The very high rate of ketone body production outstrips the body's ability to utilize them as an energy source and the blood concentration builds up. As rather strong acids, they result in a significant drop in blood pH .
Applikationshinweise The assay is linear for 1-20 nmol β-HB in up to 100 μL samples or 0.01-0.2 mM of β-HB samples.
Kommentare

Further details regarding sample type: Cell and tissue culture supernatants, urine, plasma and serum, as well as many other biological fluids

Testdauer < 1 h
Protokoll 1. Standard Curve Preparations: Dilute the beta-HB Standard to 1.0 mM by adding 10 µL of the Standard to 90 µL of distilled water, mix well. Add 0, 4, 8, 12, 16, 20 µL to a series of wells. Adjust volume to 50 µL/well with Assay Buffer to generate 0, 4, 8, 12, 16 and 20 nmol per well of the beta-HB Standard.
2. Sample Preparation: beta-HB concentrations can vary over a wide range from normal range: 20 µM-1 mM to diabetic range: 3-5 mM in serum and 10 times that in urine during diabetic ketoacidosis. Due to the presence of interfering substances in blood and urine up to about 5 µL equivalent of such samples can be tested directly. Add samples to test wells. Adjust the volume to 50 µL with beta-HB Assay Buffer To remove interfering substance from serum, serum sample can be spun filtered (10kd MWCO spin filter - The Kit ABIN413915). filtered serum can be used directly in the assay at 50 µL or up to100 µL per well. Do not use assay buffer in this case. Add enzyme mix and substrate mix as described below. For unknown samples, it may be necessary to test several different doses to ensure the readings are within the range of the standard curve.
3. Development: Mix enough reagents for the number of samples and standards to be performed: For each well, prepare a total 50 µL Reaction Mix. beta-HB Assay Buffer 46 µL beta-HB Enzyme Mix* 2 µL beta-HB Substrate Mix 2 µL Mix and add 50 µL of the Reaction Mix to each well containing beta-HB Standard or samples. *Note: Reduced pyridine nucleotides NAD(P)H can interfere with the assay. If the presence of these compounds is suspected in the sample, run a background control substituting the 2 µL Enzyme Mix with 2 µL Assay Buffer. The background reading should be subtracted from beta- HB sample reading.
4. Incubate at room temperature for 30 minutes, protect from light.
5. Measure OD at 450 nm.
6. Calculation: Correct background by subtracting the 0 beta-HB control from all standard and sample readings (Note: Reduced pyridine nucleotides NAD(P)H can interfere with the assay. If the presence of these compounds is suspected in the sample, run a background control substituting the 2 µL Enzyme Mix with 2 µL Assay Buffer. The background reading should be subtracted from beta- HB sample reading.
4. Incubate at room temperature for 30 minutes, protect from light.
5. Measure OD at 450 nm.
Ergebnisberechnung

Calculation: Correct background by subtracting the 0 beta-HB control from all standard and sample readings (The background can be significant and must be subtracted). Plot standard curve nM/well vs. standard readings. Apply sample readings to the standard curve to get the amount of beta-HB in the sample wells. The beta-HB concentration in the test samples: C = Ay/Sv (nM/µL, or μM/mL, or mM) Where: Ay is the amount of beta-HB (nmol) in your sample from the standard curve. Sv is the sample volume (µL) added to the sample well. beta-Hydroxybutyric acid molecular weight: 104.1

Beschränkungen Nur für Forschungszwecke einsetzbar
Lagerung -20 °C
Haltbarkeit 12 months
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