Troubleshooting Guide: Western Blot (2024)

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Observation: No Bands Observed
Observation: Faint Bands (Weak Signal)
Observation: Extra Bands
Observation: High Background
Observation: Diffuse Bands
Observation: White Bands (ECL method)
Observation: Patchy uneven spots all over the blot

Observation: No Bands Observed

Possible Source Suggestion
Insufficient antibody Antibody may have low affinity to protein of interest. Increase antibody concentration (2-4 fold higher than recommended starting concentration).
Antibody may have lost activity. Perform a Dot Blot.
Insufficient protein Increase the amount of total protein loaded on gel.
Confirm the presence of protein by another method.
Use a positive control (recombinant protein, cell line or treat cells to express analyte of interest).
Perform a Dot Blot.
Poor transfer Wet PVDF/Immobilon-P membrane in methanol or nitrocellulose membrane in transfer buffer.
Ensure that there is good contact between PVDF membrane and gel.
Incomplete transfer Optimize transfer time. High MW protein may require more time for transfer.
To ensure transfer is complete, stain the membrane with Ponceau S, Amido Black or India Ink.
Use prestained MW marker.
Over transfer Reduce voltage or time of transfer for low molecular weight proteins (< 10 kDa).
Isoelectric point is >9 Use alternative buffer system with higher pH such as CAPS (pH 10.5).
Incorrect secondary antibody used Confirm host species and Ig type of primary antibody.
Old antibody If antibody is expired or past manufacturer warranty, purchase fresh antibody.
Incorrect storage of antibodies Follow manufacturer's recommended storage and avoid freeze/thaw cycles.
Sodium Azide contamination Make sure buffers do not contain Sodium Azide as this can quench HRP signal.
Insufficient incubation time with primary antibody Extend incubation time to overnight at 4°C.

Observation: Faint Bands (Weak Signal)

Possible Source Suggestion
Low protein-antibody binding Reduce the number of washes to minimum.
Reduce NaCl concentration in Blotting Buffer used for wash steps (recommended range 0.15M - 0.5M).
Reduce NaCl concentration in Antibody Solution (recommended range 0.15M - 0.5M).
Insufficient antibody Antibody may have low affinity to protein of interest. Increase antibody concentration (2-4 fold higher than recommended starting concentration).
Insufficient protein Increase the amount of total protein loaded on gel.
Inactive conjugate Mix enzyme and substrate in a tube. If color does not develop or, it is weak. Make fresh or purchase new reagents. Switch to ECL.
Weak/Old ECL Purchase new ECL reagents.
Non-fat dry milk may mask some antigen Decrease milk percentage in Block and Antibody Solutions or substitute with 3% BSA.

Observation: Extra Bands

Possible Source Suggestion
Non-specific binding of primary antibody Reduce primary antibody concentration. (See Figure 1)
Reduce the amount of total protein loaded on gel.
Use monospecific or antigen affinity purified antibodies (such as R&D Systems "MAB" or "AF" designated antibodies).
Non-specific binding of secondary antibody Run a control with the secondary antibody alone (omit primary antibody). If bands develop choose an alternative Secondary Antibody.
Use monospecific or antigen affinity purified antibodies (such as R&D Systems "BAF" or "HAF" designated secondary antibodies).
Non-specific binding of primary or secondary antibodies Add 0.1 - 0.5% Tween® 20 to primary or secondary Antibody Solution.
Use 2% non-fat dry milk in Blotting Buffer as a starting point to dilute primary and secondary antibodies. Adjust antibody concentration up or down as needed. (See Figure 2a & 2b)
Increase number of washes.
Increase NaCl concentration in Blotting Buffer used for antibody dilution and wash steps (recommended range 0.15M - 0.5M). (See Figure 2c)
Increase Tween® 20 concentration in Blotting Buffer used for wash steps (0.1%-0.5%).
Aggregation of analyte Increase amount of DTT to ensure complete reducing of disulfide bonds (20 -100mM DTT). Heat in boiling water bath 5-10 minutes before loading onto gel.
Perform a brief centrifugation.
Degradation of Analyte Minimize freeze/thaw cycles of sample.
Add protease inhibitors to sample before storage.
Make fresh samples.
Contamination of reagents Check buffers for particulate or bacterial contamination. Make fresh reagents.

Observation: High Background

Possible Source Suggestion
Non-specific binding of primary antibody Use monospecific or antigen affinity-purified antibodies (such as R&D Systems "MAB" or "AF" designated antibodies).
Block in 5% milk. Adjust the milk (2-5%) or NaCl (0.15-0.5M) concentrations of primary Antibody Solution. (See Figure 3).
Decrease antibody concentration.
Non-specific binding of secondary antibody Run a control with the secondary antibody alone (omit primary antibody). If bands develop choose an alternative Secondary Antibody.
Insufficient blocking Start with 5% dry milk with 0.1%- 0.5% Tween 20, 0.15 -0.5M NaCl in 25mM Tris (pH 7.4). Incubation time may be extended. Adjust milk concentration up or down as needed.
Overnight blocking at 4°C may decrease blocking efficiency since detergents might not be effective at lower temperatures.
Non-fat dry milk may contain target antigen Substitute with 3% BSA.
Non-fat dry milk contains endogenous biotin and is incompatible with avidin/streptavidin Substitute with 3% BSA.
Some IgY antibodies may recognize milk protein Substitute with 3% BSA.
Insufficient wash Increase number of washes.
Increase Tween 20 concentration in Wash Buffer (0.1%-0.5%).
Non-Specific Binding of Primary Antibody Increase NaCl concentration in primary Antibody Solution and Blotting Buffer used for dilution of primary antibody and wash steps (recommended range 0.15M - 0.5M). (See Figure 4).
Film overexposed Reduce exposure time.
If target signal is too strong wait 5-10 minutes and re-expose to film.

Observation: Diffuse Bands

Possible Source Suggestion
Excessive protein on gel Reduce amount of protein loaded

Observation: White Bands (ECL method)

Possible Source Suggestion
Excessive signal generated Reduce antibody or protein concentration. Excessive antibody or protein can cause extremely high levels of localized signal (usually at a single band). This results in rapid, complete consumption of substrate at this point. Since there is no light production after the completion of this reaction, white bands are the result when exposed to film.

Observation: Patchy uneven spots all over the blot

Possible Source Suggestion
Contamination of reagents Check buffers for particulate or bacterial contaminate. Make fresh reagents.
Not enough solution during incubation or washing Make sure membrane is fully immersed during washes and antibody incubations.
Air bubble trapped in membrane Gently remove any air bubbles. Especially during transfer.
Uneven agitation during incubations Ensure uniform agitation by placing on a rocker/shaker.
Contaminated equipment Make sure that the electrophoresis unit is properly washed. Protein or pieces of gel remaining on the unit may stick to the membrane. Wash membrane thoroughly.
HRP aggregation Filter conjugate to remove HRP aggregates.
Long exposure Reduce exposure time.

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Possible Source	Suggestion
Insufficient antibody	Antibody may have low affinity to protein of interest. Increase antibody concentration (2-4 fold higher than recommended starting concentration).
	Antibody may have lost activity. Perform a Dot Blot.
Insufficient protein	Increase the amount of total protein loaded on gel.
	Confirm the presence of protein by another method.
	Use a positive control (recombinant protein, cell line or treat cells to express analyte of interest).
	Perform a Dot Blot.
Poor transfer	Wet PVDF/Immobilon-P membrane in methanol or nitrocellulose membrane in transfer buffer.
	Ensure that there is good contact between PVDF membrane and gel.
Incomplete transfer	Optimize transfer time. High MW protein may require more time for transfer.
	To ensure transfer is complete, stain the membrane with Ponceau S, Amido Black or India Ink.
	Use prestained MW marker.
Over transfer	Reduce voltage or time of transfer for low molecular weight proteins (< 10 kDa).
Isoelectric point is >9	Use alternative buffer system with higher pH such as CAPS (pH 10.5).
Incorrect secondary antibody used	Confirm host species and Ig type of primary antibody.
Old antibody	If antibody is expired or past manufacturer warranty, purchase fresh antibody.
Incorrect storage of antibodies	Follow manufacturer's recommended storage and avoid freeze/thaw cycles.
Sodium Azide contamination	Make sure buffers do not contain Sodium Azide as this can quench HRP signal.
Insufficient incubation time with primary antibody	Extend incubation time to overnight at 4°C.

Possible Source	Suggestion
Low protein-antibody binding	Reduce the number of washes to minimum.
	Reduce NaCl concentration in Blotting Buffer used for wash steps (recommended range 0.15M - 0.5M).
	Reduce NaCl concentration in Antibody Solution (recommended range 0.15M - 0.5M).
Insufficient antibody	Antibody may have low affinity to protein of interest. Increase antibody concentration (2-4 fold higher than recommended starting concentration).
Insufficient protein	Increase the amount of total protein loaded on gel.
Inactive conjugate	Mix enzyme and substrate in a tube. If color does not develop or, it is weak. Make fresh or purchase new reagents. Switch to ECL.
Weak/Old ECL	Purchase new ECL reagents.
Non-fat dry milk may mask some antigen	Decrease milk percentage in Block and Antibody Solutions or substitute with 3% BSA.

Possible Source	Suggestion
Non-specific binding of primary antibody	Reduce primary antibody concentration. (See Figure 1)
	Reduce the amount of total protein loaded on gel.
	Use monospecific or antigen affinity purified antibodies (such as R&D Systems "MAB" or "AF" designated antibodies).
Non-specific binding of secondary antibody	Run a control with the secondary antibody alone (omit primary antibody). If bands develop choose an alternative Secondary Antibody.
	Use monospecific or antigen affinity purified antibodies (such as R&D Systems "BAF" or "HAF" designated secondary antibodies).
Non-specific binding of primary or secondary antibodies	Add 0.1 - 0.5% Tween® 20 to primary or secondary Antibody Solution.
	Use 2% non-fat dry milk in Blotting Buffer as a starting point to dilute primary and secondary antibodies. Adjust antibody concentration up or down as needed. (See Figure 2a & 2b)
	Increase number of washes.
	Increase NaCl concentration in Blotting Buffer used for antibody dilution and wash steps (recommended range 0.15M - 0.5M). (See Figure 2c)
	Increase Tween® 20 concentration in Blotting Buffer used for wash steps (0.1%-0.5%).
Aggregation of analyte	Increase amount of DTT to ensure complete reducing of disulfide bonds (20 -100mM DTT). Heat in boiling water bath 5-10 minutes before loading onto gel.
	Perform a brief centrifugation.
Degradation of Analyte	Minimize freeze/thaw cycles of sample.
	Add protease inhibitors to sample before storage.
	Make fresh samples.
Contamination of reagents	Check buffers for particulate or bacterial contamination. Make fresh reagents.

Possible Source	Suggestion
Excessive protein on gel	Reduce amount of protein loaded

Possible Source	Suggestion
Excessive signal generated	Reduce antibody or protein concentration. Excessive antibody or protein can cause extremely high levels of localized signal (usually at a single band). This results in rapid, complete consumption of substrate at this point. Since there is no light production after the completion of this reaction, white bands are the result when exposed to film.