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Troubleshooting Guides - Western Transfer

Problem Possible Source Suggestion
Weak/No Staining Tissue Processing Increase amount of protein in sample.
Confirm protein is present through an alternate method.
  Gel ran too long Decrease running time and monitor the gel by watching dye front and ensuring the current is turned off when dye front is near bottom of gel.
  Old or degraded
protein samples
Test a fresh sample of target protein.
  Protein over-transferred/did not transfer correctly Use a membrane with a smaller pore size.
Decrease voltage for lower MW proteins (less than 10kDa). Make sure membrane is pre-soaked according to manufacturer's instructions.
Check and optimize transfer time, as appropriate transfer time will vary with MW of the target protein.
Make sure transfer "sandwich" is set up correctly and there is sufficient contact between gel and membrane. (Please see our Western Transfer protocol for correct transfer "sandwich" set up.)
Ensure the correct transfer buffer is being used.
The presence of SDS during transfer can reduce the binding of the protein when using a nitrocellulose membrane.
Add 20% methanol to transfer buffer to increase binding.
Check protein transfer by staining the membrane with a reversible stain, such as Ponceau S, to visualize major bands.
  Protein has a pI greater than 9 Substitute buffer with a higher pH buffer (i.e. CAPS buffer, pH 10.5).
  Protein masked by
blocking buffer
Test using a different blocking buffer.
Optimize concentration of protein in blocking buffer. We recommend 3% Non-Fat Dry Milk in diH2O.
  Enzyme inhibitor present
(i.e. azide)
Make sure buffers do not contain sodium azide, as this will interfere with the HRP signal.
  Old or degraded primary antibody Check manufacturer's recommendations for storage and/or expiration date. If expired or past storage time, purchase new antibody.
If antibody was subjected to repeated freeze/thaw cycles purchase new antibody, as this can affect the structure and protein:antibody binding.
Perform a Dot Blot to ensure antibody is still active.
  Insufficient amount of primary and/or secondary antibody Check manufacturer's recommendation for antibody concentration.
Test a range of concentrations to find the optimal condition for individual assay.
  Incorrect primary antibody Ensure primary antibody reacts with the target protein from the species being studied (i.e. Rabbit-anti-human
VEGF -> Human VEGF).
  Incorrect secondary antibody Ensure the secondary antibody being used is against the species of the primary antibody (i.e. Donkey-anti-rabbit
IgG -> Rabbit-anti-human VEGF).
  Insufficient incubation of primary antibody Increase incubation period of primary antibody. Antibody can be incubated for at least one hour at room temperature or overnight at 4°C.
  Insufficient incubation of secondary antibody Increase incubation period of secondary antibody (please see manufacturer's recommendations for incubation time and temperature).
  Membrane over-washed Shorten wash times and/or reduce number of washes.
Use a washing buffer that does not contain a detergent, or that has a lower concentration of detergent present.
  Incompatible developing reagent Make sure the developing reagent being used is compatible with the enzyme conjugate.
Ensure substrate is made correctly by following the manufacturer's instructions.
  Excess salt present Decrease amount of salt present in wash buffer.
Decrease amount of salt present in antibody dilution buffer.
  Degraded/Incorrect
developing reagent
Ensure the developing reagent was prepared correctly.
Check that the reagent is still active. If not, purchase
new reagent.
  For ECL method, film is bad/expired Ensure film is not expired or exposed. If so, purchase
new film.
  For ECL method, ECL developing solutions are old/expired Use fresh ECL developing solutions.
  For ECL method, film exposure time is too short Increase exposure time of the film.
  For ECL method, cling film interfering with reaction Use different brand of cling film.
High Background Antibody concentration
too high
Check manufacturer's recommendations for primary and/or secondary antibody concentrations.
Test a range of concentrations to find optimal condition for individual assay.
  Over-incubation of antibodies

Check manufacturer's protocol to ensure the correct incubation time and temperature is used. If this information is not supplied by the manufacturer, optimize incubation time through testing.

  Non-specific binding of antibodies

Ensure the primary antibody is specific only for the protein that is being targeted.
Run a control, omitting the primary antibody incubation. If bands develop, choose a different secondary antibody.
Choose a different host species for secondary antibody.

  Cross reaction of antibodies with proteins in blocking buffer and/or sample Use a different blocking buffer formulation.
Goat antibodies will react with BSA due to a slight recognition of the bovine protein by the goat antibody. This will lead to a purple sheen on your membrane. If this occurs, use a different species antibody or a different blocking buffer.
If secondary antibody is suspected, dilute antibody in buffer containing 1-5% normal serum from the same species as target protein.
  Ineffective blocking buffer Use a different blocking buffer formulation.
Add detergent to blocking buffer, such as TWEEN-20.
  Insufficient blocking Use a different blocking buffer formulation.
Batch of blocking buffer may be inactive. Use fresh blocking buffer.
If blocking overnight at 4°C, this may decrease blocking efficiency since lower temperatures may lessen the effectiveness. Try incubation for one hour at room temperature.
Add TWEEN-20 to blocking buffer.
Optimize incubation time and/or temperature of
blocking step.
  Insufficient washing After each step, wash membrane 3 times for 10 minutes each. Increase the duration of the washing step if this is not sufficient.
Try a different detergent in washing buffer or add TWEEN-20 if not already present.
Increase volume of washing buffer used.
  Over-incubation in color development substrate Shorten incubation time in color development solution. Monitor development until bands are present and stop reaction by washing membrane in diH2O.
  Excess enzyme present Reduce concentration of selected enzyme conjugate.
  Interference by
endogenous enzyme
Non Fat Dry Milk contains endogenous biotin and may interfere with avidin/biotin detection system. Use different blocking buffer, such as 3% BSA.
  Contaminated reagents Filter buffers before use to remove contaminant.
Make fresh buffers and re-run Western Transfer.
  Membrane problems Test new membranes.
During the entire procedure the membrane can only be dried between transfer and immunostaining. Once immunostaining of the membrane has begun, the membrane cannot be dried until completion of immunostaining procedure. Re-run Western Transfer if membrane was dried at any other point during immunostaining procedure.
  For ECL method, ECL film overexposed Shorten film exposure time.
If signal from target protein is too strong, wait 5-10 minutes and re-expose to film.
Splotchy background Uneven agitation during incubations Ensure the membrane is agitated evenly by placing on a rocker or shaker during incubation.
  Insufficient amount of incubation buffer Ensure enough solution is present during each incubation period to fully submerge the membrane and allow it to float freely in the solution.
  Air bubbles present
during transfer
Gently remove all air bubbles from transfer "sandwich" before transfer is started. This can be done by using a roller or clean glass rod/Pasteur pipette.
  Membrane problems Ensure membrane is wetted thoroughly according to the manufacturer's protocol.
Handle membranes with extreme care. Mishandled membranes can cause non-specific binding.
Handling membranes with bare hands can lead to contamination of membrane. Use forceps or wear gloves when handling membrane.
  Aggregation of HRP conjugate If using HRP, filter conjugate to remove HRP aggregates.
Use a fresh sample of HRP conjugate.
  Contamination of reagents Filter buffers before use to remove contaminant.
Make fresh buffers and re-run Western Transfer.
Non-specific bands Antibody concentration
too high
Check manufacturer's recommendations for primary and/or secondary antibody concentrations.
Test a range of concentrations to find optimal condition for individual assay.
  Old or degraded antibody Check manufacturer's recommendations for storage and/or expiration date. If expired or past storage time, purchase new antibody. If antibody was subjected to repeated freeze/thaw cycles purchase new antibody, as this can affect the structure and protein:antibody binding.
  Non-specific binding of primary antibody Decrease concentration of primary antibody.
Check concentrations of protein being added to gel.
Use a more specific antibody, such as a monoclonal antibody or Antigen Affinity Purified polyclonal antibody.
Add a small amount of detergent, such as TWEEN-20 (0.1-0.5%) to the primary antibody solution and/or blotting buffer.
Increase number/duration of washes.
  Non-specific binding of secondary antibody Decrease concentration of secondary antibody.
Run a control, omitting the primary antibody incubation. If bands develop, choose a different secondary antibody.
Add a small amount of detergent, such as TWEEN-20 (0.1-0.5%) to the secondary antibody solution.
Increase number/duration of washes.
  Aggregation of target protein Add or increase a reducing agent, such as DTT or BME, to ensure all disulfide bonds are reduced.
Heat protein sample in a hot water bath for 5-10 minutes before loading onto gel.
Check concentration of protein being added to gel. A concentration that is too high can cause protein aggregation. Re-run Western Transfer with a lower concentration of protein.
  Degradation of protein sample Make fresh samples and minimize freeze/thaw cycles
of samples.
Add protease inhibitor to samples prior to storage.
Keep samples stored between -20°C and -80°C.
  Insufficient blocking Test a different blocking buffer formulation.
Batch of blocking buffer may be inactive. Use a fresh blocking buffer.
If blocking overnight at 4°C, this may decrease blocking efficiency since lower temperatures may lessen the effectiveness. Try incubation for one hour at room temperature.
Add TWEEN-20 to blocking buffer.
Optimize incubation time and/or temperature of
blocking step.
  SDS facilitates binding to immobilized protein bands Ensure membrane is sufficiently washed after transfer
of proteins.
Do not use SDS during immunostaining when using a nitrocellulose membrane.
  Excess enzyme present Reduce concentration of the selected enzyme conjugate.
  Contamination of reagents Filter buffers before use to remove contaminant.
Make fresh buffers and re-run Western Transfer.
Diffused/Fuzzy bands Antibody concentration
too high
Check manufacturer's recommendations for primary and/or secondary antibody concentrations.
Test a range of concentrations to find optimal condition for individual assay.
  Excess protein on gel Further dilute protein samples before loading onto gel.
  Gel over-heated/high voltage Heat causes the gel to lose its structure and resolving power. Be sure the transfer reservoir is run in an ice bath.
Reduce voltage or current used during transfer.
  Incorrect preparation of membrane Pre-soak membrane in correct transfer solution (see membrane manufacturer's protocol). Ensure it is soaked for the correct amount of time.
White Bands
(ECL visualization method)
Antibody or protein concentration too high/Excessive
signal generated
White bands are the result of rapid consumption of the substrate caused by the presence of too much conjugate. This is the result of high concentrations of the antibodies and/or proteins. Check manufacturer's recommendations for antibody and/or protein concentrations.