Introduction:Formaldehyde (Methanal) is the simplest aldehyde. It is widely employed in industry for a wide range of applications. Formaldehyde is also used as a disinfectant and is a commonly utilized tissue fixative and embalming agent. Formaldehyde is naturally present in all tissues and body fluids. Recently it has been shown that some cancer types exhibit elevated formaldehyde levels. Increased formaldehyde concentration in urine has been associated with prostate and bladder cancer. Thus, measuring formaldehyde in urine can be a very useful tool when studying cancer.
This Formaldehyde Assay Kit provides a convenient fluorimetric means to measure formaldehyde in biological samples. In the assay, formaldehyde is derivatized with acetoacetanilide in the presence of ammonia. The resulting fluorescent product is then quantified fluorimetrically excitation/emission wavelength: 370/470nm). The assay is simple, sensitive, stable and high-throughput adaptable. The assay can detect as low as 1.5uM formaldehyde in biological samples.
Key Features:Safe: Non-radioactive assaySensitive and accurate: As low as 1.5uM (45ppb) formaldehyde can be quantified
Homogenous and convenient: "Mix-incubate-measure" type assay. No wash and reagent transfer steps are involved. Robust and amenable to HTS: Can be readily automated on HTS liquid handling systems for processing thousands of samples per day.
Applications:Formaldehyde determination in urine and other biological samples.
Kit Contents:F6000-03A: Reagent A, 1x5mlF6000-03B: Reagent B, 1x3mlF6000-03C: Formaldehyde Standard, 1x100ulF6000-03D: 10% TCA, 1x5mlF6000-03E: Neutralizer, 2 x 1.5ml
Storage and Stability:The kit is shipped at room temperature. Store all reagents at 4°C. Shelf life of 12 months after receipt.
Materials Required But Not Provided:Pipeting devicesCentrifuge tubesBlack flat bottom 96-well plates (e.g., Corning Costar)Plate reader
Precautions:Reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.
Assay Procedure:Note: Use black flat-bottom plates. Prior to assay, bring all reagents to room temperature.
1. StandardsMix 5ul of the provided formaldehyde standad with 495ul dH2O to make a 100uM Premix.
Transfer 50ul standards into separate wells of the plate.
2. Sample PreparationUrine samples should be diluted 2.5-fold with dH2O. If urine samples contain visible particulates, then the samples should be cleared by either filtration or centrifugation (14,000 rpm, 5 min.).
Samples high in protein (cell lysate, serum, etc.) need to be deproteinated and neutralized prior to assaying. To deproteinate, add 50ul 10% TCA per 100ul sample. Vortex and centrifuge for 5 min at 14,000 rpm. Transfer 100ul of clear supernatant to a clean tube and neutralize with 25ul Neutralizer. Note: Measured ∆RFU’s for deproteinated samples need to be multiplied by 1.875 to compensate for the resulting dilution of the sample. Samples not measured the same day should be stored frozen, preferably at -80°C.
3. Add 50ul of each prepared sample to two separate wells of the plate (one well will be used as a Sample Blank).
4. Prepare Working Reagent for each standard and sample well by mixing 33ul Reagent A and 22ul Reagent B. For the Sample Blanks, make the following Working Reagent: 33ul Reagent A + 22ul dH2O. Add 50ul of the appropriate Working Reagent to each well. Tap plate to mix. Incubate at room temperature for 30 min protected from light.
5. Read fluorescence intensity using 370nm excitation wavelength and 470nm emission wavelength.
Calculations:Plot the RFU measured at 30 min for each standard against the standard concentrations. Determine the slope using linear regression fitting. The formaldehyde concentration of a sample is calculated as follows:[Formaldehyde] = [(RFU sample - RFU blank - RFU water)/Slope] x n
where:RFU sample, RFU blank and RFU water are the measured fluorescence values of the sample, sample blank and water, respectivelySlope is the slope of the standard curve in uM-1n is the dilution factor (n= 1.875 for deproteinated samples).
The calculated concentration is expressed in uM unit.
Note: If the sample formaldehyde concentration is higher than the 100uM prior to applying the dilution factor, dilute sample in water and repeat the assay. Multiply result by the dilution factor.
Conversion Factor: 1uM formaldehyde is equivalent to 300 ppb.
