Troubleshooting

Store reagents properly

Read the data sheet carefully and store reagents in their proper location. Some kits require different components to be stored at different temperatures.

Equilibrate reagents to assay temperature

Use an incubator or water bath to equilibrate all reagents (except enzymes) to assay temperature. Enzymes should be thawed and kept on ice or at 4°C during assays.

Run a test standard curve

Make sure that there is nothing faulty with the reagents and to ensure that you are following the directions carefully. Your standard curve should be linear unless otherwise noted in data sheet and generate similar results to the example graph.

Preliminary sample serial dilution

If this is your first time running a particular type of sample, use micro-centrifuge tubes to make a 2x serial dilution of your sample. Run the assay with the original and diluted samples to determine if dilution is necessary and which dilution factor is optimal. Use the chosen dilution factor in all subsequent experiments.

Pipette carefully

Consistency between wells is very important; carefully pipette down the side of the well and ensure that all wells have the same volume and all reagents flow down to the bottom. Take special care to avoid bubbles, which can disrupt absorbance readings.

Assay not working (no signal)

Possible cause: Assay buffer is too cold, causing low enzyme activity
Solutions: Equilibrate reagents to room temperature or specified assay temperature.

Possible cause: Omission of a reagent or step in the protocol
Solutions: Re-read the data sheet and follow instructions carefully.

Possible cause: Plate read at incorrect wavelength
Solutions: Re-read the data sheet and follow instructions carefully.

Possible cause: Use of an incorrect microplate
Solutions: Absorbance: clear plates; Fluorescence: black plates (bottoms can be clear or black); Luminescence: white plates.

Samples with peculiar signals

Possible cause: Incompatible sample type
Solutions: Re-read the data sheet for list of compatible samples.

Possible cause: Improper sample preparation
Solutions: For enzyme activity assays, do not deproteinate your sample. Deproteination kills all enzyme activity. If you do need to deproteinate, make sure that you have neutralized your sample properly. EDTA is a metal chelator, and interferes with a few of our assays. Any assay that measures metal ions or requires a metal cofactor is incompatible with EDTA.

Signals are too high

Possible cause: Standards were made too concentrated, saturating the signal
Solutions: Refer back to the data sheet and ensure that standard dilutions are done correctly.

Possible cause: Working reagent was made incorrectly, saturating the signal
Solutions: Refer back to the data sheet and ensure that working reagent was made correctly.

Possible cause: Samples are too concentrated
Solutions: Dilute your samples and repeat the experiment.

Signals are too low

Possible cause: Reagents incorrectly stored
Solutions: Check that reagents have been properly stored. They may degrade rapidly if incorrectly stored.

Possible cause: Reagents have expired
Solutions: Check the shelf life of the kit.

Possible cause: Samples are too dilute
Solutions: Concentrate or remake your samples with more cells/tissue.

Signals jump up and down

Possible cause: Unmixed and non-uniform wells
Solutions: Tap plate a few times quickly to mix contents thoroughly.

Possible cause: Bubbles in the wells
Solutions: Check wells for air bubbles. Repeat assay and pipette carefully avoiding bubbles.

Possible cause: Precipitate in the wells
Solutions: Check well for precipitates or turbidity. Determine cause of precipitation and dilute, deproteinate, or use some other alternative sample treatment to eliminate precipitation.

Standard curve is not linear

Possible cause: Pipetting errors and variability
Solutions: Remake standard dilutions and closely follow instructions while carefully pipetting.

Possible cause: Incorrect calculations
Solutions: Re-check the calculations. Some assays have different equations.

Possible cause: Standard curve maybe a non-linear fit
Solutions: Refer to the data sheet for the fitting equation and example graph.

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