Troubleshooting

General Recommendations

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.

Troubleshooting

• Assay Not Working (No Signal)

POSSIBLE CAUSES	SOLUTIONS
Assay buffer is too cold, causing low enzyme activity	Equilibrate reagents to room temperature or specified assay temperature.
Omission of a reagent or step in the protocol	Re-read the data sheet and follow instructions carefully.
Plate read at incorrect wavelength	Re-read the data sheet and follow instructions carefully.
Use of an incorrect microplate	Absorbance: clear plates; Fluorescence: black plates (bottoms can be clear or black); Luminescence: white plates.

• Samples With Peculiar Signals

POSSIBLE CAUSES	SOLUTIONS
Incompatible sample type	Re-read the data sheet for list of compatible samples.
Improper sample preparation	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 CAUSES	SOLUTIONS
Standards were made too concentrated, saturating the signal	Refer back to the data sheet and ensure that standard dilutions are done correctly.
Working reagent was made incorrectly, saturating the signal	Refer back to the data sheet and ensure that working reagent was made correctly.
Samples are too concentrated	Dilute your samples and repeat the experiment.

• Signals Are Too Low

POSSIBLE CAUSES	SOLUTIONS
Reagents incorrectly stored	Check that reagents have been properly stored. They may degrade rapidly if incorrectly stored.
Reagents have expired	Check the shelf life of the kit.
Samples are too dilute	Concentrate or remake your samples with more cells/tissue.

• Signals Jump Up and Down

POSSIBLE CAUSES	SOLUTIONS
Unmixed and non-uniform wells	Tap plate a few times quickly to mix contents thoroughly.
Bubbles in the wells	Check wells for air bubbles. Repeat assay and pipette carefully avoiding bubbles.
Precipitate in the wells	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 CAUSES	SOLUTIONS
Pipetting errors and variability	Remake standard dilutions and closely follow instructions while carefully pipetting.
Incorrect calculations	Re-check the calculations. Some assays have different equations.
Standard curve maybe a non-linear fit	Refer to the data sheet for the fitting equation and example graph.