Troubleshooting

ELISA Kits

Things to Note Before Beginning Experiment
  • 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.
The standard curve is poor, and overall OD values are low

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.
The standard curve is normal, but sample OD values are low

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.
High background

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 37oC.
Low sensitivity

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.)
No signal from entire plate

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.
Poor linearity

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.
Large discrepancies in experimental results, artifacts, or false positives

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.

Antibodies

Western Blot: No Band or Weak Band

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
Western Blot: High Background

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
Western Blot: Non-Specific Bands

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
Western Blot: Other Experimental Problems
  • 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
Immunohistochemistry: Sample is Unstained

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
Immunohistochemistry: Weak Positive Stain

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
Immunohistochemistry: Non-Specific Staining

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
Immunofluorescence: Weak or No Fluorescence

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
Immunofluorescence: High Background Fluorescence

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
Immunofluorescence: Fast Fluorescence Quenching

Possible Reasons:

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

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
Flow Cytometry: Weak or No Fluorescence

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
Flow Cytometry: High Background Fluorescence

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
Flow Cytometry: Abnormal Fluorescence

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

Molecular

Dual (Column/Reagent) Kits: DNA Isolation

Problem: Low DNA yield

  • Possible Cause: incomplete sample lysis
  • Possible Cause: Absolute ethanol not added to Buffer W2 before use
  • Possible Cause: Ethanol not added to lysate before adding to column
  • Possible Cause: pH of Buffer E is too low, or Buffer E has not been preheated
  • Possible Cause: Poor quality starting material or sample was not stored appropriately

Problem: DNA has degraded

  • Possible Cause: Sample was not fresh or stored appropriately, or was subjected to freeze-thaw cycles
  • Possible Cause: DNase contamination

Problem: Inhibition of downstream enzymatic reactions

  • Possible Cause: Purified DNA contains residual ethanol
  • Possible Cause: Purified DNA contains residual salt
Column Kits: DNA Isolation

Problem: Low DNA yield

  • Possible Cause: incomplete lysis
  • Possible Cause: Ethanol not added to Buffer W2 before use
  • Possible Cause: Incorrect elution conditions
  • Possible Cause: Poor quality starting materials or inappropriate storage conditions

Problem: DNA has degraded

  • Possible Cause: Sample is not fresh or has been subjected to repeated freeze/thaw cycles
  • Possible cause: DNase contamination
  • Possible Cause: Excessive pipetting or homogenization has damaged the DNA

Problem: RNA contamination

  • Possible Cause: incomplete removal of RNA (use RNase treatment)

Problem: Inhibition of downstream enzymatic reactions

  • Possible Cause: Purified DNA contains residual ethanol
Column Kits: DNA Isolation for PCR Products

Problem: Low DNA yield

  • Possible Cause: Ethanol not added to Buffer W2 before use
  • Possible Cause: Nuclease contamination
  • Possible Cause: Column is overloaded; decrease the loading volume or starting sample amount
  • Possible Cause: Gel has not completely dissolved; increase gel extraction time
  • Possible Cause: incorrect elution conditions
  • Possible Cause: Volume of elution buffer used is too small

Problem: Inhibition of downstream enzymatic reactions

  • Possible Cause: Incorrect buffer used to elute DNA
  • Possible Cause: Purified plasmid contains residual ethanol

Problem: DNA passes through in the flow-through or wash fraction

  • Possible Cause: Column is overloaded; decrease the loading volume or starting sample amount
  • Possible Cause: Inappropriate salt or pH conditions in buffers used

Problem: Purified DNA floats out of wells while running in agarose gels

  • Possible Cause: Traces of ethanol not fully removed from column before eluting product
Column Kits: RNA Isolation

Problem: Low RNA yield

  • Possible Cause: incomplete lysis or homogenization
  • Possible Cause: Incorrect elution conditions

Problem: Low integrity or degraded RNA

  • Possible Cause: RNase contamination

Problem: Inhibition of downstream enzymatic reactions

  • Possible Cause: Purified RNA contains residual ethanol
Column Kits: Viral Nucleic Acid Isolation

Problem: Low yield

  • Possible Cause: incomplete lysis
  • Possible Cause: Residual buffer from previous steps present during the elution step
  • Possible Cause: Viral nucleic acid stuck on column (repeat elution step and incubate the column for 5 min with water prior to centrifugation)

Problem: Degraded RNA

  • Possible Cause: Poor starting material quality or inappropriate storage
  • Possible Cause: RNase contamination

Poor RNA performance in downstream applications

  • Possible Cause: purified product contains residual ethanol
Magnetic Bead Kits: DNA Isolation

Problem: DNA is sheared or degraded

  • Possible Cause: lysate mixed too vigorously with pipette
  • Possible Cause: DNase contamination
  • Possible Cause: water (pH>7) present in elution/release step

Problem: RNA contamination

  • Possible Cause: incomplete removal of RNA (use RNase treatment)

Problem: low DNA yields

  • Possible Cause: Incomplete lysis and/or homogenizationPossible Cause: incorrect preparation of magnetic beads (vortex tube and fully resuspend beads before use)
  • Possible Cause: Protein contamination (use Proteinase K treatment)
  • Possible Cause: Incorrect elution/release conditions
  • Possible Cause: poor starting material quality or inappropriate storage

Problem: High background on UV measurement

  • Possible Cause: residual beads present in purified product (repeat magnetic separation if necessary)
Plasmid Kits: Plasmid Isolation

Problem: RNA contamination

  • Possible Cause: incomplete removal of RNA (use RNase treatment)

Problem: Plasmid bands smear on agarose gel

  • Possible Cause: Plasmid DNA degradation (keep preparations on ice or frozen)

Problem: Poor plasmid quality

  • Possible Cause: Genomic DNA contamination; do not overgrow bacterial cultures

Problem: Low DNA yield

  • Possible Cause: Low plasmid copy number
  • Possible Cause: Ethanol not added to Buffer W2 before use
  • Possible Cause: Nuclease contamination
  • Possible Cause: Column overloaded
  • Possible Cause: the SDS in Buffer S2/M2 has precipitated in storage (to resolve, incubate at 30-40oC and mix well)
  • Possible Cause: Incorrect elution conditions
  • Possible Cause: Plasmid lost in e. coli host

Problem: Inhibition of downstream enzymatic reactions

  • Possible Cause: Incorrect buffer used to elute DNA
  • Possible Cause: Purified plasmid contains residual ethanol

Problem: DNA passes through in the flow-through or wash fraction

  • Possible Cause: Column is overloaded; decrease the loading volume or starting sample amount
  • Possible Cause: Inappropriate salt or pH conditions in buffers used

Problem: Plasmid DNA floats out of wells while running in agarose gels

  • Possible Cause: Traces of ethanol not fully removed from column before eluting product
Reagent Kits: DNA Isolation

Problem: Difficult to dissolve DNA

  • Possible Cause: incomplete removal of ethanol
  • Possible Cause: pellet has been overdried

Problem: RNA contamination

  • Possible cause: incomplete removal of RNA (Use RNase treatment)

Problem: Low DNA yield

  • Possible Cause: incomplete lysis and/or homogenization
  • Suggestion: Grind tissue samples thoroughly, and ensure you are using the appropriate method for lysate preparation based on the amount of starting materials. Cut tissue samples into smaller pieces and ensure the tissue is completely immersed in lysis buffer
  • Possible Cause: Presence of ethanol
  • Possible Cause: separation phase was not repeated until interphase is clear
  • Possible Cause: Incorrect Precipitation conditions or precipitation time was too short

Problem: DNA has degraded

  • Possible Cause: Sample used was not fresh or was not stored appropriately
  • Possible Cause: DNase contamination

Problem: Inhibition of downstream enzymatic reactions

  • Possible Cause: Presence of ethanol in purified DNA

Problem: A260/280 ratio is below ~1.7

  • Possible Cause: Incomplete removal of proteins or other contaminants
Reagent Kits: RNA Isolation

Problem: Difficult to dissolve RNA

  • Possible Cause: incomplete removal of all ethanol

Problem: Genomic DNA contamination

  • Possible Cause: incomplete removal of gDNA (use DNase treatment)

Problem: Degraded or low integrity RNA

  • Possible Cause: RNase contamination (consider RNase inhibitor)

Problem: Low RNA yield

  • Possible Cause: incomplete lysis and/or homogenization
  • Suggestion: Grind tissue samples thoroughly, and ensure you are using the appropriate method for lysate preparation based on the amount of starting materials. Cut tissue samples into smaller pieces and ensure the tissue is completely immersed in lysis buffer
  • Possible Cause: Incorrect precipitation conditions
  • Possible Cause: Poor starting material quality or storage

Problem: Inhibition of downstream enzymatic reactions

  • Possible Cause: Presence of ethanol in purified RNA

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