Troubleshooting

• All reagents in the kit should be stored according to the manual instructions.
• Before beginning the experiment, all components should be brought to room temperature.
• To ensure the uniformity and accuracy of each reagent, mix all components before use.
• Keep the temperature stable at room temperature throughout the entire experiment.
• The pH should be maintained between 7.2-7.4 for the entire duration of the experiment.
• Ensure all concentrated reagents are appropriately diluted prior to beginning the experiment.
• Washing technique should be kept consistent throughout the experiment and done according to the instructions in the manual.
• Avoid creating bubbles in solutions to ensure uniformity of all reactions.
• Take the reading of the microtiter plate 2 minutes after adding the stop solution.

Possible reasons:

• Improper dilution of the standard and/or sample. Always pay close attention to properly dilute the standard and any samples.
• Insufficient dissolution of the standard. Pay careful attention to ensure thorough mixing and complete dissolution of the freeze-dried powder in the standard vial(s).
• Insufficient reaction time, or low environmental temperature. Follow the reaction time and incubation temperature recommended in the paper instruction manual included in the kit and preheat the incubator in advance.

Possible reasons:

• The protein content of the target protein in the sample is below the detection range. If the target protein concentration is just below the detect limit, try increasing the sample volume. If the sample OD value is very low, consider switching to a more sensitive ELISA kit.
• Improper storage of samples, leading to sample degradation or contamination. After sample collection, avoid repeated freeze-thaw cycles and use samples as soon as possible.

Possible reasons:

• Inadequate washing. Wash all wells thoroughly according to the recommended wash procedure in the paper instruction manual. If background remains high, try increasing the number of washes.
• Contamination of the washing solution. Use fresh washing solution, clean glassware, and change pipette tips to avoid contamination.
• Failure to terminate the reaction in a timely manner. Pay close attention to the reaction time after adding the substrate, observe color change midway, and add the stop solution when time is up.
• Deterioration or contamination of the substrate. Always use fresh substrate, and examine before the experiment to see if it is contaminated or has turned blue (substrate should be colourless). If you notice any issues, request a replacement bottle before beginning your experiment. During the substrate incubation, avoid light exposure.
• Failure to read results in a timely manner. Preheat the microplate reader in advance, and read the results promptly after adding the stop solution.
• Contaminated or dirty glassware. Ensure all instruments, glassware, and consumables are clean before the experiment.
• Excessively high incubation temperature. Ensure the incubator temperature is consistent and maintained at 37^oC.

Possible reasons:

• Sample(s) are a poor fit for use with the kit. Understand your sample’s background and origin in relation to the specifications of the kit.
• The kit is not sensitive enough for the samples. If the target protein content in the sample is inherently low or has a low expression level, we recommend using a more sensitive assay kit that better matches your sample concentration.
• The kit has expired. Always check the expiry date of the kit before using.
• The sample diluent is contaminated with a strong acid or base, or the pH of the experiment is compromised (too high or low).
• The target protein concentration in the sample is too high, resulting in the hook effect. (The hook effect is when falsely low results are produced from an immunoassay in the presence of excess target analyte. The excess analyte interferes with antibody binding and results in fewer enzyme complexes being formed, artificially lowering the results.)

Possible Reasons:

• Detection Reagent/Solution B has degraded. We recommend mixing 1 uL of prepared Detection Reagent B or Detection Solution B and add 50uL of substrate to see if there is a color change. If there is no color change, it indicates that the Detection Reagent/Solution B’s chromogenic agent is ineffective.
• Solutions used in the wrong order, or otherwise mixed up. Ensure that you are using the correct solution, in the correct order as described in the procedure. Some users mistakenly use the washing solution as a diluent, resulting in a blank plate. Other users may mix up the order of Detection Reagent/Solution A and Detection Reagent/Solution B, resulting in a blank plate.
• Inappropriate reagents used. Always use only the reagents provided in the kit. Do not mix reagents from other kits or other lots together, as this can cause enzyme contamination or inactivation.
• The pH of the experiment is incorrect. Avoid introducing any reagents that might alter the pH of the experiment.

Possible reasons:

• Blockage of some liquid dispensing nozzles in the plate washer, resulting in uneven washing and potentially skipped wells.
• Cross-contamination during manual tapping of the plate. Try to reduce any splashing when removing liquid from the wells.
• Cross-contamination during sample addition. Always wear protective equipment and change pipette tips as needed to avoid any contamination.
• Failure to equilibrate all reagents and samples to room temperature before the experiment.
• Insufficient dissolution of freeze-dried powder during the preparation of the standard solutions.

Possible reasons:

• Sample factors. Improper collection and handling of specimens, severe hemolysis or lipemic samples, and the presence of red blood cells or fibrinogen in the serum can impact the results. These substances easily precipitate or adhere to the polyethylene wells of the ELISA plate, making them difficult to wash away.
• For samples with hemolysis, turbidity, or presence of clots, the sample should be allowed to naturally coagulate and shrink for 1-2 hours, or be placed in a 37 degree Celsius water bath for 10 minutes. Appropriate centrifugation speed should be used, and centrifugation time should be extended; for example, centrifuging at 3000r/min for 15 minutes to ensure sufficient precipitation of blood cells and fibrinogen. After separation of blood cells and serum, the supernatant can be collected and used. Efforts should be made to minimize the false positive rate caused by non-specific substances in the sample by washing thoroughly and diluting samples appropriately.
• Procedural factors. Cross-contamination during sample addition can often lead to false positives. Take care to store the plate and perform the experiment in a clean environment away from any contamination or sample residue. Take care when adding samples to the wells to add it directly to the center of the well to prevent any non-specific binding in the upper walls of the wells.
• Washing procedure. It is important to thoroughly and evenly wash all wells at each wash step in the procedure to ensure even binding of the desired antibodies and enzymes, and to wash away any impurities and non-specific proteins. Always perform all recommended washing steps and try to keep all washing steps as consistent as possible. Have the same user perform all washing steps, as well as reagent addition steps, for maximum consistency. 
• Substrate degradation. Ensure that the substrate is colourless and clear of any contamination before use.
• Edge Effects. Edge effects refer to a deeper colouration in the outer wells as compared to the central wells in a 96-well plate. This is due to the different thermodynamic gradients on the surfaces of peripheral and central wells of the plate. An uneven working environment temperature can cause this. We recommend using a water bath, or when adding the reaction solution to the plate, heat both the plate and the solution to a water bath temperature (e.g., 37 degrees Celsius) to avoid edge effects and improve measurement reproducibility.
• Human Factors. Always follow the procedure outlined in the manual as closely as possible for best results. The same user should perform the entire experiment to ensure consistency, especially with the washing procedures. Failure to timely replace pipette tips during the experiment, changing any experimental steps, extending or shortening reaction times, arbitrarily increasing or decreasing the number of washes or the volume of wash buffer, or inaccurate preparation of the wash buffer, can result in unexpected or inaccurate experimental results.

• Additional materials are often needed but not supplied in the kit contents. This includes common laboratory equipment and reagents. Ensure that you have all necessary materials and reagents before beginning the experiment.
• The absorbance wavelength to set the microplate reader at varies depending on the kit. Please ensure that you set your microplate reader to the wavelength specified in the manual.
• Reagents included in the kit often need to be prepared before use in the experiment. Read the manual carefully and follow all preparation steps before beginning.
• Depending on the samples used with the kit, some optimization may be necessary. A preliminary experiment may be necessary to ensure that the results fall within the standard curve range and can be measured accurately.

• The method of calculating results differs between kits. Please follow the instructions found in the manual or contact us with any questions or for additional assistance.
• The results calculated are closely correlated to the operation of the experiment and the experimental environment. Please follow all instructions found in the paper manual as closely as possible for best results.

Possible reasons:

• Solutions used in the wrong order, or otherwise mixed up. Ensure that you are using the correct solution, in the correct order as described in the procedure.
• Inappropriate reagents used. Always use only the reagents provided in the kit. Do not mix reagents from other kits or other lots together, as this can cause contamination or inactivation.
• The pH of the experiment is incorrect. Avoid introducing any reagents that might alter the pH of the experiment.

Possible reasons:

• Enzyme activity or analyte concentration is too low to be detected accurately. Try using lower dilutions of samples or increase starting sample amount during preparation.
• Improper storage or handling of samples, leading to degradation. Follow sample preparation instructions in the manual for best results.
• Insufficient reaction time, or low environmental temperature. Follow the reaction time and incubation temperature recommended in the paper instruction manual included in the kit and preheat any instruments in advance.

Possible reasons:

• Contamination of solutions. Use freshly prepared solutions, clean glassware, and change pipette tips to avoid contamination.
• Failure to read results in a timely manner. Preheat the microplate reader in advance, and read the results promptly when indicated in the manual.
• Deterioration or contamination of the substrate, dye reagents, or other important reagents. (Included reagents vary between kits.) Always use freshly prepared solutions and examine them before the experiment. If you notice any issues, request a replacement before beginning your experiment.

Possible reasons:

• Sample factors. Varied samples, or different or improper handling and preparation of samples can lead to differences in the results.
• Procedural factors. Cross-contamination during sample addition can lead to false positives or otherwise unexpected results. Take care to perform the experiment in a clean environment away from any contamination or sample residue. Take care when adding reagents to the wells to add them directly to the center of the well to mix properly with the other reagents.
• Human Factors. Always follow the procedure outlined in the manual as closely as possible for best results. The same user should perform the entire experiment to ensure consistency. Failure to timely replace pipette tips during the experiment, changing any experimental steps, extending or shortening reaction times, or other changes can result in unexpected or inaccurate experimental results.

Possible Reasons:

• Not enough sample was loaded in the well
• The amount of target protein is too low or is not expressed in the sample; consider using a positive control sample
• Poor transfer of target protein to membrane
• Antibody concentration is too low
• Antibody concentration is too high
• Not enough exposure time
• The substrate has been deactivated, or has not been incubated long enough
• Target protein was degraded or denatured during transfer; consider lowering the transfer temperature, decreasing the current or transfer time
• Membrane washing issues; consider reducing the frequency or duration of washing
• Antibody used is inactive or not concentrated enough

Possible Reasons:

• Experimental equipment is contaminated
• Membrane material is contributing to background
• Blocking buffer is interfering or cross-reacting with the antibodies
• Blocking step is not long enough
• Antibody concentration is too high
• Membrane washing issues; ensure there is no contamination and the washing step is long enough
• Exposure time is too long
• Other contamination during the experiment

Possible Reasons:

• Protein sample was digested during the experiment
• Too much sample was loaded in the well
• Blocking step is not long enough
• Washing step is not long enough
• Antibody concentration is too high
• Antibody specificity is too low
• The target protein used has multiple spliceosomes or modified sites

• Black spots on membrane – Blocking buffer is not suitable and may have non-specifically bound with the antibodies used
• White bands on membrane – Target protein or antibody concentrations are too high
• Molecular weight is not as expected – Gel is not mixed properly, or the concentration is incorrect
• Uneven or migrating bands – Equipment is not working properly; sample is not dissolved thoroughly; electrodes are not balanced; or too much sample was loaded

Possible Reasons:

• One or more steps have not been completed in the procedure
• Secondary antibody is not compatible with detection method used
• Primary and secondary antibody are not compatible
• Target protein is not expressed or not expressed enough in the sample
• Antibody concentration is not high enough
• One or both antibodies used has degraded
• Substrate has degraded
• pH of buffers used is not compatible with the experiment

Possible Reasons:

• Poor antigen retrieval; using heat-induced epitope retrieval and/or enzymatic digestion is recommended
• Too much liquid left on experimental area
• Area/section was not securely horizontal during incubation
• Proper fixation method was not used

Possible Reasons:

• Deparaffinization time was insufficient
• Endogenous enzymes/biotin were present
• Blocking time was not long enough
• Blocking buffer used was not a good match
• Antibody specificity was too low
• The washing step is not long enough
• Primary or secondary antibody concentration is too high
• DAB incubation time is too long
• Paraffin or cells have dried out
• Unwanted cross-reactivity with endogenous proteins and secondary antibody
• Antigen translocation due to sample treatment used

Possible Reasons:

• Target protein is not expressed or not expressed enough in the sample
• Antigen epitope was affected by immobilization
• Poor cell permeability
• Antigen was lost due to cell permeability; consider decreasing concentration or time of permeability reagent used
• Primary or secondary antibody concentration is too low
• Poor secondary antibody choice

Possible Reasons:

• Primary antibody is not specific enough
• Primary or secondary antibody concentration is too high
• Blocking time is insufficient
• IgG found in blocking buffer used
• Washing protocol is insufficient
• Cells have dried out
• Antigen was lost due to cell permeability; consider decreasing concentration or time of permeability reagent used
• Fluorescence microscope was not used with appropriate settings

Possible Reasons:

• The fluorescein-conjugated secondary antibody used has poor photo stability
• A sealing agent to prevent quenching was not used

Possible Reasons:

• Any auto-fluorescence was not quenched after fixing cells
• Some part of the sample exhibits auto-fluorescence; consider using a negative control and reducing background of the microscope
• Cell components present in the sample exhibit auto-fluorescence
• The amount of dead to living cells in the sample is too high

Possible Reasons:

• Improper storage or handling of antibodies used
• Antibody fluorescence was quenched before experiment
• cells used exhibit high auto-fluorescence similar to antibodies used
• Ineffective dye time or temperature
• Inappropriate procedure used for intracellular protein staining
• Attempted detection of extracellular secretory proteins
• Target protein has very low expression in sample used; consider using a brighter dye for better detection
• Antigens have been damaged by sample preparation or storage
• Improper functioning of flow cytometer laser; check calibration
• Inappropriate filters used for data collection
• Overcompensation of data
• Inappropriate cell gates used
• Poor data analysis

Possible Reasons:

• Cells used exhibit high auto-fluorescence
• Antibodies used have bound to dead cells; consider using active dyes
• Inappropriate use of Cy5 and other blue dyes
• Antibody concentration is too high
• Cells were dyed for too long
• Washing procedure was insufficient
• Experimental compensation regulation was insufficient

Possible Reasons:

• Incorrect concentration of isotype control used
• Poor match of isotype control and detection antibodies
• Adhesion or dead cells were included
• Experimental conditions have affected cell characteristics