Troubleshooting Tips

WB
- Old detection reagent or substrate kits
  
  Use a new detection buffer.
- Sodium azide inhibition
  
  Avoid using buffers containing sodium azide, which may quench HRP signals.
- Insufficient incubation with the primary antibody
  
  Increase the incubation period with the primary antibody or perform overnight incubation at 4℃.
- Over-wash the membrane
  
  Do not over-wash the membrane - excessive washing may wash away the primary antibody. Try washing three times for 5 minutes.
- Blocking buffer
  
  Non-fat milk may mask antigens. Try to reduce the amount of milk in the blocking buffer and antibody solution or substitute it with different blocking reagents.
- Incomplete transfer
  
  Optimize the transfer conditions (e.g. time, electrical current). Reassure the completion of the transfer procedure before moving on to the next step
- Over-use of antibodies
  
  Use fresh antibody solution for the best performance.
- Incompatible secondary antibody used
  
  Use a secondary antibody that was rasied against the host species of the primary antibody. Use positive controls to ensure that the secondary antibody is working properly.
- Insufficient primary antibody
  
  Use higher concentrations of the antibody. Our protocol provides a guideline for antibody dilutions. Use positive controls to ensure that the primary antibody is working properly.
- Difficult target proteins are presented
  
  Try to use the most sensitive detection reagent.
- Insufficient protein loaded into the gel
  
  Increase the amount of sample for gel loading. Use 25-50ug cell/tissue lysate protein per lane. Load at least 25ug cell lysate protein per lane.
- Overexposure
  
  Decrease the exposure time.
- Insufficient washing
  
  Increase the number of washes with PBS to remove unwanted residual antibodies.
- Insufficient blocking
  
  Extend the blocking time or perform overnight blocking at 4℃.
- Cross-reaction between blocking reagent and antibodies
  
  Add Tween 20 (detergent) to the washing buffer or try a different blocking buffer.
- Non-specific binding of the antibody
  
  Increase the blocking incubation period. Add 5% non-fat milk powder and Tween 20 to the blocking buffer.
- The concentration of antibodies is too high
  
  Dilute the antibody concentration further or incubate it shorter.
- Protein degardation or digested
  
  Use protease inhibitors to improve the condition.
- Different splice variants
  
  Search information related to the expression of the target protein.
- Protein modification
  
  Search related documents or use some bioinformatic tools for modification prediction.
- The secondary antibody concentration is too high
  
  Decrease the incubation period or the concentration of the antibody. Run a lane with the secondary antibody only as a control.
IHC
- Incompatible secondary antibody
  
  Choose a secondary antibody that is against the IgG of the host species in which the primary antibody has been raised.
- Insufficient incubation time with antibodies
  
  Extend the incubation time.
- Insufficient amount of antibody for the protein detection
  
  Try to use higher concentrations of the primary antibody with a longer incubation time
- Insufficient amount of target protein present in the sample
  
  Check information related to the tissue specificity of the target protein. Use the most sensitive detection reagent.
- Masked antigen
  
  Use appropriate antigen retrieval method to break the protein cross-links formed by formalin fixation.
- Insufficient deparaffinization
  
  Refresh the xylene solution and deparaffinize the sample sections for a longer period.
- Autofluorescence or natural fluorescence existed in tissue
  
  Before incubating with the antibody, check the tissue slide with a fluorescence microscope. If the fluorescence signal is detected, choose other detection systems, not the fluorescent method.
- Endogenous biotin signal
  
  Pretreat the tissue sample with unconjugated avidin if the biotin-avidin detection system is used.
- Endogenous alkaline phosphatase activity
  
  Pretreat the tissue sample with levamisole solution if AP is used as the label.
- Endogenous peroxidase activity
  
  Pretreat the tissue sample with 3% H2O2 before incubating it with the primary antibody.
- Non-specific binding of the secondary antibody
  
  Run a lane of control with only the secondary antibody.
- The concentration of the antibody may be too high
  
  Decrease the antibody concentration.
ELISA
- Over incubation with the substrate
  
  Try decreasing the incubation time to prevent signal over-development.
- Substrate incubation is performed under light
  
  After adding substrate, put the plate in the dark immediately.
- Reused plates
  
  Use a new plate.
- The substrate buffer is old
  
  Use fresh substrate reagent.
- The antibody concentration may be too high
  
  Decrease the concentration of the antibody.
- Insufficient blocking time
  
  Extend the blocking time or use more/different blocking solutions.
- Contamination of the negative control
  
  May be contaminated by other samples or reagents. Avoid overflowing among wells. Pipetting carefully.
- Insufficient amount of washing for the plates
  
  Make sure wells are filled up with wash buffer and ensure the complete removal of the residual solution from each well. Increase the number of washes.
- Incorrect wavelength
  
  Make sure that the correct wavelength is set to measure the signal.
- Insufficient incubation time for the substrate solutions
  
  Optimize the incubation time until the plate has developed enough for the ELISA reader.
- Insufficient antibody used
  
  Try to use more concentrated antibody. Incubate the antibody for the recommended amount of time
- The target protein is not expressed in the sample
  
  Search information related to the expression profile of the target protein. Increase the amount of sample used or use more sensitive detection reagents.
- Pipetting error
  
  Be careful upon sample loading and avoid the presence of bubbles in wells.
- Uneven coating
  
  Check the coating procedure and use appropriate ELISA plates.
IF
- Sample fixation destroys epitope
  
  Try a different sample fixation method such as formalin or glutaraldehyde. Methanol may cause the cell membrane to collapse.
- Cell permeability is poor
  
  Use 0.1 Triton X-100 for 15 min to permeabilize cells or increase the time of incubation.
- Insufficient wash
  
  The slides should be gently washed using PBS. Do not immerse in wash buffer for long periods or douse with distilled water.
- The secondary antibody was exposed to light
  
  Store the secondary antibody in the dark.
- Insufficient secondary antibody used
  
  Make sure the secondary antibody is specific to the primary antibody.
- Blocking is too strong
  
  Reduce blocking time.
- Too few cells expressing the protein
  
  Try to use more cells in the sample or use a stably transfected cell line.
- Protein is not expressed in cells
  
  Prepare a cell extract and check that the target protein is present in cells by Western blots.
- Antibody incubation time is too short
  
  Extend the incubation time.
- Antibody concentration is too diluted
  
  A higher concentration is suggested.
- The sample was washed not enough
  
  Wash thoroughly with PBS at the end of each step for 5 minutes. Repeat the process 5 times.
- The incubation temperature is too high
  
  Incubate samples at 4℃ overnight or at room temperature for 4 hours.
- The secondary antibody is not specific
  
  Check that the secondary antibody is binding specifically. Run a secondary control without a primary antibody.
- The incubation period is too long
  
  Incubate with primary/ secondary antibody for a short time.
- The primary antibody is too concentrated
  
  Dilute the antibody concentration.
- Incorrect blocking buffers used
  
  Try to use a different blocking buffer such as 5% goat serum in PBS for 1 hour.
- Over fixation of the test sample
  
  Decrease fixation time.