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.
Poor Standard Curve

Possible reasons:

  • Degradation of the standard due to time or improper storage
  • Standard curve dilutions were prepared incorrectly
  • Pipetting errors leading to inconsistent concentrations
  • Standard curve data was plotted improperly or without using appropriate software
No Signal

Possible reasons:

  • The reagents have expired - check packaging for expiration date
  • Adding detection reagent A, B or TMB substrate was skipped
  • The distilled water used for dilution was contaminated
  • An enzyme inhibitor (such as Sodium azide) is present
  • Wash buffer is too concentrated and has not been appropriately diluted
  • The labelled enzyme is contaminated or inactive
  • pH value of solution is too high or low. All solutions should be maintained at pH 7.2-7.4 for the entire experiment
Weak Signal

Possible reasons:

  • The reagents have expired or have not been stored in the correct conditions - check packaging for expiry date
  • The reagents, standard, and/or sample were not warmed to room temperature prior to conducting the experiment
  • Incorrect amounts and/or dilutions of reagents were used
  • One or more solutions used were contaminated
  • The labelled enzyme is contaminated or inactive
  • TMB substrate was not incubated long enough
  • Washing procedure problems, i.e.:
    • Wash buffer too concentrated
    • Wells were washed too many times
    • Wells were washed too vigorously
    • Wells were washed too long each time
No Colour Gradient

Possible reasons:

  • TMB substrate was stored incorrectly (not in a cool dark place and protected from light exposure)
  • The incubation time was too long (can lead to strong, nonspecific adsorption)
  • Wells were not washed adequately at one or more steps
  • Cross contamination from pipette tips or other contaminated reagents
  • Low concentration or low activation of the coated or detection antibody
Poor Replicate Data

Possible Reasons:

  • Wells were washed inconsistently or insufficiently at one or more steps 
  • Reagents were mixed or prepared inconsistently 
  • One or more solutions were contaminated
  • Bubbles formed in wells

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

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