ChIP Troubleshooting Guide 3

  1. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    Not enough cells/chromatin

    Add enough chromatin for each IP experiment. We suggest using at least 25 μg of chromatin for each IP.

    Incorrect Protein A/G/L used

    Make sure that the Protein A/G/L beads are capable of binding to the antibody subclass being used.

    Cross-linking process too long

    Over Cross-linking with formaldehyde might mask the antibody binding sites and reduce antibody binding ability. It is advisable to optimize the cross-linking steps by using different concentration of formaldehyde or reducing cross-linking time.

    Not enough antibody

    Titre antibody amount used for each IP to determine the optimal condition. Up to 10ug of antibody can be used for each IP experiment.


     

    Washes too stringent

    Reduce the number of washes. Reduce salt concentration in the wash buffer.


     

    Antibody not capable of
    immunoprecipitation

    Try a different antibody. Try polyclonal antibody if monoclonal antibody does not work well.


     

    The chromatin size might be too small

    Make sure that the shearing condition is not too harsh which might results in fragments of DNA smaller than what the primers are able to amplify.


     

    Incomplete elution from the Protein
    A/G/L beads

    Incubate beads in elution buffer at 65°C with frequent mixing.

  2. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    PCR reagent might be contaminated

    Prepare new solutions from stock.

  3. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    Inadequate washing

    Use a more stringent washing buffer. Try to  use a high salt washing buffer or increase the number of washes.

    Non specific binding to Protein A,G or L

    Include a pre-clear step by incubating lysate with Protein A/G/L agarose beads.

    Too much DNA template added
    to the PCR reaction, or too many
    cycles of amplification

    Add less DNA template or reduce the number of cycles of amplification.  Alternatively, real-time PCR can be used for the detection of ChIPed DNA products.

    Buffers may be contaminated

    Use freshly prepared lysis or wash buffers.

IP Troubleshooting Guide 3

  1. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    Insufficient antibody

    Check the recommended amount of antibody as indicated in the datasheet.  Titrate and optimize the optimal antibody amount used per IP experiment.

    Washes too stringent

    Reduce the number of washes. Reduce salt concentration in the wash buffer.

    Incorrect Protein A/G/L used

    Make sure that the Protein A/G/L beads are capable of binding to the antibody subclass being used.

    Target protein not present in the
    sample used

    Make sure that the target protein is expressed at a relatively high level in the sample used by including an Input sample in the WB.

    Incorrect Lysis buffer used

    Make sure that the lysis buffer used is not over-denaturing and destroy the native conformation of the target proteins.

    Antibody not capable of
    immunoprecipitation

    Try a different antibody. Try polyclonal antibody if monoclonal antibody does not work well.

  2. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    Secondary antibody rcognizes heavy / light chain denatured from primary 
    antibody

    Use secondary antibodies which only recognizes native form of IgG for immunoblotting.

  3. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    Inadequate washing

    Use a more stringent washing buffer. Try to  use a high salt washing buffer or add 0.2% SDS or 1% Tween20 to washing buffer. Increase the number of washes.

    Concentration of antibodies too high

    Check the recommended amount of antibody as indicated in the datasheet.  Titrate and optimize the optimal antibody amount used per IP experiment.

    Non specific binding to Protein A,G or L

    Pre-block beads with BSA. Incubate beads with 2%BSA in PBS for 1 hour wash in PBS before use.

    Non specific binding to agarose beads

    Include a pre-clear step by incubating lysate with Protein A/G/L agarose beads.

    Antibody not specific enough

    Use affinity purified and pre-absored antibody for IP experiments.

    Sample degradation

    Add adequate protease inhibitors and phosphatase inhibitors throughout sample preparation and Ip steps.

Western Blot Troubleshooting Guide 7

  1. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    Protein post-translationally modified or alternatively spliced

    Check if the protein of interest is post-translationally modified or alternatively spliced and produce other isoforms.

    Incomplete protein denaturation

    Freshly add DTT or β-Mercaptoethanol to sample buffer, or adequaltely boil samples to ensure complete bond breakage between peptides.

    Membrane protein issue

    If a membrane protein is to be detected, try low temperature (~65°C) or avoid boiling which might cause aggregation of membrane proteins.

  2. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    Protein post-translationally modified or alternatively spliced

    Mix gel completely before pouring. If SDS-PAGE should be run the day after preparation, make sure that the gel is prepared at RT and store in moist chamber at 4°C. 

    Protein degradation

    Keep the voltage and temperature low while running SDS-PAGE. If necessary, run gel in the cold room. Remove bubbles trapped at the bottom of gel to ensure even electrophoresis.

    Protein Multimerization

    Make sure that the salt concentration of lysis buffer is kept between 0.15M to 0.5M.

    Antibody concentration too high

    Make sure that total amount of protein loaded into each well is between 20-50μg.

    Antibody issue

    Optimize the dilution factor of primary and secondary antibody.

    Interference from secondary antibody

    While performing Immunoprecippitaiotn (IP) experiment, make sure that the secondary antibody used to detect the protein of interest is derived from a species different from that of antibody used to pull down protein. Alternatively, use a secondary antibody that recognize only the native form of IgG to detect IP protein.

  3. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    Reagent contaminated

    Prepare all reagents freshly.

    Blocking agent insufficiently dissolved

    Make sure that the blocking agent such as milk or BSA is completely dissolved before use. Alternatively, filter the blocking solution with 0.45μm filter before use.

  4. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    Poor gel preparation

    Mix gel completely before pouring. If SDS-PAGE should be run the day after preparation, make sure that the gel is prepared at RT and store in moist chamber at 4°C. 

    Voltage, temperature too high, field effect

    Keep the voltage and temperature low while running SDS-PAGE. If necessary, run gel in the cold room. Remove bubbles trapped at the bottom of gel to ensure even electrophoresis.

    High salt concentration in samples

    Make sure that the salt concentration of lysis buffer is kept between 0.15M to 0.5M.

    Protein overloaded

    Make sure that total amount of protein loaded into each well is between 20-50μg.

    Antibody concentration too high

    Optimize the dilution factor of primary and secondary antibody.

  5. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    Poor gel preparation

    Mix gel completely before pouring. If SDS-PAGE should be run the day after preparation, make sure that the gel is prepared at RT and stored in moist chamber at 4°C. 

    Protein overloaded

    Make sure that total amount of protein loaded into each well is between 20-50μg.

    High membrane protein concentration in samples

    If membrane fraction is used, make sure that sample is sufficiently diluted before loading into SDS-PAGE.

ELISA Troubleshooting Guide 5

  1. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    Improper standard dilution

    Use appropriate diluent as blank. Make sure that the dilution is performed as according to protocol

    Standard improperly reconstituted

    Briefly spin standard vial before opening. Make sure that there is no undissolved material after reconstituting

    Standard degraded

    Store standards as according to protocol

    Curve doesn’t fit the scale

    Try plotting log-log or 5 parameter logistic curve fit

    Pipetting error

    Calibrate pipettes to make sure that the correct volume is dispensed

    Incomplete washing

    Increase washing cycles

  2. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    Improper washing

    Make sure that the washing is done as according to protocol

    Poor mixing of samples

    Mix samples gently and evenly

    Dirty plate

    Make sure that the bottom of plate is clean

    Reagents too old

    Make sure that the reagents are not expired. Use freshly prepared reagents

    Bubbles in wells

    Make sure that there is no bubble in wells before reading

    Inconsistent pipetting

    Calibrate pipettes to make sure that the correct volume is dispensed

    Edge effects

    Make sure that the plate and reagents are equilibrated to room temperature before starting assay

  3. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    Too much antibodies was used

    Reduce the concentration of primary or secondary antibodies

    Antibodies bind nonspecifically

    Use blocking buffer or choose another affinity-purified antibody

    Too much substrate reagent used

    Use substrate with higher dilution

    Insufficient washing

    Increase washing cycles

    Wrong concentration of blocking reagent

    Check the recommended concentration of blocking buffer

    Reaction not stopped

    Stop reactions with STOP buffer before reading

    Plate left too long before reading

    Take measurements shortly after addition of substrate and STOP buffer

    Insufficient Tween in wash buffer

    Use PBS+0.05% Tween as wash buffer

    Incubation temperature too high

    Optimize incubation temperature for each experiment

    Plate stacking during incubation lead to uneven temperature throughout the plate

    Avoid stacking plates together during incubation

    Pipetting error

    Calibrate pipettes to make sure that the correct volume is dispensed

    Reagents not mixed properly

    Make sure that all reagents are mixed properly and equilibrated to room temperature before assay

    Salt concentration of incubation and wash buffer

    Increase salt concentration to reduce nonspecific interaction

    Substrate incubation carried out in light

    Perform substrate incubation in dark

    Dirty plate

    Make sure that the bottom of plate is clean

  4. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    Insufficient coating

    Use more antigens or antibodies for coating

    Substrate reagents have expired or prepared at a wrong pH

    Use fresh substrate reagents

  5. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    Assay set up incorrectly

    Make sure that the instructions in the protocol is followed carefully

    Incorrect secondary antibody used

    Check if the correct secondary antibody is used

    Insufficient antibodies used

    Increase concentration of primary or secondary antibody

    Substrate reagents not fresh

    Use fresh substrate reagents

    Wrong settings of plate reader

    Check the settings (wavelength, filters, gain etc) of plate reader

    Insufficient incubation

    Follow the incubation time as indicated in the protocol booklet

    Sample concentration falls below detection limits of kit

    Decrease dilution factor or concentrate samples

    Plate washing too vigorous

    Check the setting of plate washer. Pipette wash buffer into wells gently

    Wells dried out

    Cover plate with adhesive film or incubate in humidified chamber throughout experiment

    Enzyme inhibitor present in buffers or reagents

    Inhibitors such as Sodium Azide can affect enzyme and assay performance. Ensure that there is no enzyme inhibitor in any buffers

Immunohistochemistry Troubleshooting Guide 3

  1. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    Insufficient blocking

    Select appropriate serum as blocking buffer. Blocking for 1 hour at room temperature.

    Interference from endogenous enzymes

    Perform H2O2 or Levamisole quenching.

    Non-specific binding from primary antibodies

    Dilute primary or secondary antibody. Choose another IHC-validated primary antibodies.

    Inadequate washing

    Increase washing cycles/time. Increase salt/detergent concentration
    for stronger washes.

    Non-specific binding from secondary antibodies

    Perform secondary antibody incubation only. Use pre-adsorbed secondary antibody.

    Non-specific binding from chromogen

    Perform chromogen incubation only. Use other chromogen if necessary.

    Interference from secondary antibody in multicolor staining

    Make sure that the fluorochrome does not overlap with one another.

    Autofluorescence issue

    Make sure that there is no endogenous background caused by tissue itself. Check under fluorescence microscope prior to staining to identify autofluorescence.

  2. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    Antigen retrieval too harsh

    Optimize retrieval steps to give the best morphology.

    Tissue sections peeled off slide

    Dry samples for 2-4 hours at 60°C.
    Tissue with high lipid content (eg breast tissues) should be dried for longer time.

    Sectioning issue

    Cut thinner slides for better resolution: 3-5 μm. Use a new/sharper blade.

    Autolysis has occurred

    Fix samples as soon as possible.
    Choose other fixatives to accelerate penetration.
    Fixative perfusion might be necessary for larger tissues.

  3. Check the possible causes and find out the solution!

     

    Possible Causes

    What You Can Do?

    Over fixation

    Reduce fixation time. Perform antien retrieval to unmask epitopes.

    Insufficient fixation

    Increase fixation time or try other fixative.

    Fixation process delayed

    Fix immediately as tissue is extracted.

    Permeabilization issue

    For nuclear/cytoplasmic proteins, add permeabilization agent (eg Triton X-100, Saponin) in blocking and antibody incubation buffer. 
    For membrane/tight junction proteins, avoid permeabilization agent.

    Primary antibodies not suitable for IHC

    Choose an IHC-validated primary antibodies.

    Wrong secondary antibody used

    Make sure that primary and secondary antibodies match one another.

    Low expression of protein in tissue samples

    Use signal amplification methods (eg: HRP Polymer ARG80982, ARG80967, ARG80966)

    Insufficient deparafinization

    Make sure that parafin is removed completely before staining.