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Palmitic Acid Standards for Colorimetric DetectionAdd 0, 2,

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Palmitic Acid Standards for Colorimetric DetectionAdd 0, 2, 4, 6, 8, and 10 mL of the standard solution intoa 96 well plate, generating? (blank), 2, 4, 6, 8, and 10 nmole/well standards. Add Fatty Acid Assay Buffer to each well to bring the volume to 50 mL.Tissue (10 mg) or cells (1 ?106) can be homogenized in 200 mL of a 1% (w/v) Triton X-100 in chloroform solution. Centrifuge the samples at 13,000 ?g for10 minutes to remove insoluble material. Collect the organic phases (lower phase) and air dry at 50 ? to remove chloroform. Vacuum dry for 30 minutes to remove trace chloroform. Dissolve?he dried?ipids in 200 mL of Fatty Acid Assay Buffer by vortexing extensively for 5 minutes. The solution may be turbid or cloudy.?erum and other liquid samples can be directly added to wells.?ote: For unknown samples, it is suggested to test several sample dilutions to ensure the readings are within the linear range of the standard curve.?ring samples to a final volume of 50 mL with Fatty Acid Assay Buffer. Assay Reaction1.??Add 2 mL of ACS Reagent to each sample and standard well, and incubate for 30 minutesat 37 ?.?.??Set up the Master Reaction Mix according to the scheme in Table 1. 50 mL of the Master Reaction Mix is required for each reaction (well).?able 1.Master Reaction Mix?eagent Volume Fatty Acid Assay Buffer 44 mL Fatty Acid Probe 2 mL Enzyme Mix 2 mL Enhancer 2 mL?.??Add 50 mL of the Master Reaction Mix to each of the wells. Mix well using a horizontal shaker or by pipetting, and incubate the reaction for 30 minutes at 37 ?. Protect the plate from light during the incubation.?.??For colorimetric assays, measure the absorbance at 570 nm (A570). For fluorometric assays, measure fluorescence intensity (lex = 535/lem = 590 nm).?esultsCalculationsThe background for either assay is the value obtained for the 0 (blank) palmitic acid standard. Correct for the background by subtracting the blank value from all readings. Background values can be significant and must be subtracted from all readings.?se the values obtained from the appropriate palmitic acid standards to plot a standard curve. The amount of fatty acids present in the samples may be determined from the standard curve.Note: A new standard curve must be set up each time the assay is run.?oncentration of Fatty Acids Fa/Sv = CFa = Amount of Fatty Acids in unknown sample (nmole)from standard curveSv = Sample volume (mL) added to reaction well C = Concentration of Fatty Acids in sampleFFA Assay (Sigma CAT No: MAK044) ProtocolWIL Assay Standards/Sample OD values, as per protocol PALM STDOD @ 570nm OD @ 570nm0 0.0510.0482 0.3710.36640.7140.7116 1.1021.09781.422 1.41310 1.711.721Patient ID ( C) 0.4330.439The concentration from the standard curve was found to be 2.312 n mole /wellquestions: 1- calculate to find the concentration in Patient Tissue (100mg) was homogenised in 2ml (extracted as per protocol), and dissolved in 2ml of Free Fatty Acid Assay Buffer before adding 50ml to each well?2- How does the Assay work ?3- senesitivity /Range ,will the assay detect the concentration of molecule in the volume of assayed sample and is the read outproportional to the amount of molecul;e present in the analyzed sample??iologyScienceBiochemistryMETABOLISM 300848

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