Western Blot Troubleshooting Guide

The Western Blot technique , also known as an immunoblot, is an immunoassay commonly used to identify, by means of antibodies , proteins previously separated by electrophoresis.

In this post we bring you a short guide with tips for solving problems in Western Blot , based on the most common causes.

How To Troubleshoot Western Blot

The most frequent problems usually translate into one or more of the following aspects: high background noise, weak or non-existent signal, multiple or diffuse bands, uneven staining of the gel and / or irregular and uneven spots throughout the blot.

Below we detail the possible causes (PC) as well as the tips or solutions (S) to solve them:

1.-High Background Noise :

PC: Antibody concentration too high

  • S: Optimize and reduce antibody concentration

PC: Formation of aggregates of the secondary antibody

  • S: Filter the secondary antibody or use a new one

PC: Antibody incubation temperature too high

  • S: Incubate the antibody at 4ºC

PC: Nonspecific binding of the secondary antibody or cross-reactivity with the blocking agent

  • S1: Run control of secondary antibody (without primary)
  • S2: Reduce the concentration of the secondary antibody

PC: Cross reactivity of primary or secondary antibody with blocking agent

  • S: Add Tween-20 to the incubation and wash buffers

PC: Incompatible blocking agent

  • S: Compare different blocking buffers

PC: Incomplete crash

  • S1: Optimize the choice of the blocking buffer
  • S2: Increase the protein concentration in the blocking buffer
  • S3: Optimize blocking time and / or temperature
  • S4: Add Tween-20 to the blocking agent

PC: Insufficient lock

  • S: Extend the blocking time or use a compatible blocking agent

PC: Cross reactivity of the antibody with other proteins

  • S1: Use different blocking agents
  • S2: Reduce the concentration of the secondary antibody
  • S3: Analyze the cross reactivity between the secondary antibody and the membrane

PC: insufficient washing

  • S1: Increase the number of washes and the volume of the buffer
  • S2: Add Tween-20 to the wash buffer

PC: Exposure time too long

  • S: Reduce exposure time

PC: Problems with the membrane

  • S1: Use a new membrane
  • S2: Make sure the membrane stays moist
  • S3: Handle carefully avoiding damage to the membrane

PC: Incompatible membrane

  • S: In general, nitrocellulose membranes tend to give less background noise

PC: Contaminated buffer

  • S: Filter the buffer or use a new one

2.- Weak Or Non-Existent Signal

PC: Incorrect transfer of protein to membrane

  • S1: Use Ponceau to check the transfer efficiency
  • S2: Ensure the correct contact between the membrane and the gel during the transfer
  • S3: Moisten the membrane according to the instructions
  • S4: Avoid overheating during transfer
  • S5: Use positive controls or molecular weight markers
  • S6: Optimize transfer time and current

PC: Insufficient bond between protein and membrane

  • S: Add methanol to the transfer buffer

PC: Insufficient antibody

  • S: Increase antibody concentration

PC: Insufficient antigen

  • S: Load more protein

PC: Masking of antigen by blocking buffer

  • S1: Compare different blocking buffers
  • S2: Optimize protein concentration of blocking buffer
  • S3: Reduce blocking time

PC: Presence of sodium azide in buffers

  • S: Remove sodium azide from buffers

PC: Exposure time too short

  • S: Increase the exposure time

PC: Substrate incubation time too short

  • S: Increase incubation time

PC: Digestion of the protein in the membrane

  • S: Optimize the amount of blocking agent

PC: Degradation of protein during storage

  • S: Re-prepare the sample with the protein

PC: Incompatibility between primary and secondary antibody

  • S1: Make sure compatibility
  • S2: Use loading controls for Western Blot to check the effectiveness of the secondary antibody (You can consult the guide on “How to select loading controls for western blot” here )

PC: Low concentration of primary and / or secondary antibody

  • S1: Increase the concentration of antibody
  • S2: Increase incubation time

PC: Cross reactivity between blocking agents and antibodies

  • S1: Add Tween 20
  • S2: Change the blocking agent

PC: Inability of the primary antibody to recognize the protein

  • S1: Review the instructions for use
  • S2: Use positive controls

PC: Not enough protein

  • S1: Increase the amount of charge in the gel
  • S2: Use protease inhibitors
  • S3: Use positive controls

PC: Insufficient transfer and excessive washing

  • S1: Confirm the transfer with Ponceau
  • S2: Avoid excessive washing

PC: excess lock

  • S1: Change the blocking agent
  • S2: Reduce blocking time

PC: Inhibition of the secondary antibody by sodium azide

  • S: Avoid co-use of sodium azide with HRP-conjugated antibodies

PC: Methanol concentration too high

  • S: Reduce the concentration of methanol or use isopropyl alcohol

PC: Idle Conjugate

  • S: Mix the enzyme and the substrate in a tube to confirm that a colorimetric reaction occurs

3.- Multiple Bands

PC: The protein in the sample has been digested

  • S1: Make sure that enough protease inhibitor has been added to the buffer
  • S2: Avoid sample freeze / thaw cycles

PC: Nonspecific binding of the primary antibody

  • S1: Reduce the concentration of primary antibody
  • S2: Reduce the amount of protein loaded in the gel
  • S3: Purify the antibody by affinity with the antigen

PC: Too much protein per lane or too sensitive detection system

  • S: Make serial dilutions of the starting material

PC: Ineffective lock

  • S: Modify blocking conditions

PC: Antigen concentration too low

  • S: Try to enrich the antigen by fractionation or immunoprecipitation

PC: Nonspecific binding of the secondary antibody

  • S1: Run a secondary antibody control, without the primary
  • S2: Select another secondary antibody (You can consult the guide to select secondary antibodies here )
  • S3: Adjust the antibody concentration
  • S4: Increase the number of washes
  • S5: Use monospecific antibodies

PC: Analyte forms aggregates

  • S1: Increase the amount of DTT
  • S2: Heat in a water bath before loading the gel
  • S3: Centrifuge briefly

PC: Contamination of reagents

  • S1: Check that the buffers do not contain particles or microbial contamination
  • S2: Use new reagents

4.- Fuzzy Bands

PC: Excess protein in the gel

  • S: Reduce protein load

5.- Uneven Staining Of The Gel

PC: Bacterial contamination

  • S: Keep the antibodies at 4ºC and use fresh buffers (you can consult the guide on “How to store the antibodies” here )

PC: Insufficient volume of antibody

  • S: Make sure the membrane is covered by the antibody and shake during incubation

6.- Irregular And Uneven Points Throughout The Blot

PC: Contamination of reagents

  • S1: Check that the buffers do not contain particles or bacterial contamination
  • S2: Use new reagents

PC: Insufficient volume of solution during incubation or washing

  • S: Make sure that the membrane is completely immersed during the washes and incubations with the antibodies

PC: Air bubbles trapped in the membrane

  • S: Eliminate bubbles, especially during transfer

PC: Irregular shaking during incubation

  • S: Make sure the stirring is uniform

PC: HRP Aggregation

  • S: Filter conjugates to remove aggregates

Western Blot is a technique that involves several steps, and there are no universal optimal conditions that can be applied to all samples. For this reason, there are a multitude of variables that may affect the results of the tests as expected.

We hope that these tips have been useful for solving problems in Western Blot . And if you still have any questions, do not hesitate to contact us.

Glenda Montgomery

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