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:

• 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

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

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

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

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

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