Western Blot Troubleshooting

In this western blot troubleshooting section, we will help you visually identify specific and common problems on your western blots, such as high background, weak or no signal, multiple bands, uneven staining and suggest what may be causing them and some solutions to remedy them.

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Protein gel electrophoresis troubleshooting

Electrophoresis troubleshooting- bands lose resolution, are not straight

Protein bands lose resolution, lanes have streaks and are not straight

Possible cause	Solutions
Too much protein loaded per lane	Reduce the sample loads. The maximum recommended sample load for optimal resolution in mini gels with 10, 12, 15, or 17 wells is 0.5 μg per band or about 10–15 μg of cell lysate per lane.

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Protein electrophoresis troubleshooting, streaks at lane edges, dumbbell shaped bands

Viscous samples, streaks at sample lane edges, dumbbell-shaped bands, lane widening

Possible cause	Solutions
Excess salt (ammonium sulfate) in sample during gel electrophoresis	Perform dialysis to decrease salt concentration. Use a small dialysis device such as the Thermo Scientific Slide-A-Lyzer MINI Dialysis Device, 0.5 mL.
	Concentrate and resuspend samples in lower-salt buffer prior to electrophoresis. Use small-volume concentrators such as Thermo Scientific Pierce Protein Concentrators PES, 0.5 mL.
	Make sure that the salt concentration does not exceed 100 mM.

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Protein electrophoresis troubleshooting, narrowing lanes, undefined bands

Protein aggregation resulting in narrow lanes that cannot be interpreted

Possible cause	Solutions
DNA contamination— genomic DNA in the cell lysate may cause the sample to become viscous, resulting in protein aggregation, which can affect protein migration patterns and resolution	Shear genomic DNA to reduce viscosity before loading the sample.

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Protein electrophoresis troubleshooting, lane widening

Uneven sample lanes, lane widening

Possible cause	Solutions
Excess salt (sodium chloride) in sample during gel electrophoresis. High salt concentrations result in increased conductivity, which affects protein migration and can result in protein bands spreading into adjacent lanes containing samples with normal salt concentrations	Perform dialysis to decrease salt concentration. Use a small dialysis device such as the Slide-A-Lyzer MINI Dialysis Device, 0.5 mL.
	Concentrate and resuspend samples in lower-salt buffer prior to electrophoresis. Use small-volume concentrators such as Pierce Protein Concentrators PES, 0.5 mL.
	Make sure that the salt concentration does not exceed 100 mM.
High detergent concentration (e.g., SDS or Triton X-100 detergent) in gel electrophoresis. Detergents form mixed micelles with the anionic detergent SDS in the gel and migrate down into the gel; they interfere with the SDS–protein binding equilibrium	Most of the nonionic detergents (e.g., Triton X-100, NP-40, and Tween 20 detergents) interfere with SDS- polyacrylamide gel electrophoresis (SDS-PAGE). Keep the ratio of SDS to nonionic detergent at 10:1 or greater to minimize these effects.
	Use detergent removal columns or the Thermo Scientific Pierce SDS-PAGE Sample Prep Kit to remove excess detergent.
High concentration of RIPA (radioimmunoprecipitation assay) buffer results in widening of lanes and significant streaking during electrophoresis	Dilute samples before electrophoresis to lower the final concentration of lysis buffer to prevent buffer-related defects.

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Shadow at lane edges

Possible cause	Solutions
Excess reducing agent in the lysis or sample buffer	The final concentration of reducing agents for SDS-PAGE should be less than 50 mM for DTT (dithiothreitol) and TCEP (tris(2-carboxyethyl) phosphine), and less than 2.5% for β-ME (β-mercaptoethanol).

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General western blot troubleshooting

Western blot troubleshooting, nonspecific bands

Nonspecific or diffuse bands

Possible cause	Solutions
Antibody concentration too high	Reduce concentrations of antibodies, particularly of primary antibody.
Too much protein loaded on gel	Reduce the amount of sample loaded on gel.
Signal from chemiluminescent substrate too strong	Reduce the length of time the blot is exposed to film.
	Reduce the concentration of the substrate.
	Shorten incubation time of membrane with substrate.
	Completely remove substrate after incubation period.
	Decrease the concentration of antibodies, particularly HRP- and AP-conjugated antibodies.

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High background

Possible cause	Solutions
Antibody concentration too high	Decrease concentration of primary and/or secondary antibody.
Incompatible blocking buffer	Do not use milk with avidin–biotin system. Milk contains biotin, which will result in high background.
	When probing for phosphoproteins, avoid phosphate- based buffers like PBS and phosphoprotein-containing blockers like milk or casein. Instead, block with BSA in Tris-buffered saline.
	Test for cross-reactivity in blocking buffer by blocking a clean piece of membrane, incubating with antibodies, and then detecting with the substrate of choice.
	When using an alkaline phosphatase (AP) conjugate, a blocking buffer in Tris-buffered saline (TBS) should be selected because phosphate-buffered saline (PBS) interferes with AP activity.
	Try a different blocking buffer. Use our blocking buffer selection guide to find the most compatible blocking buffer for your experiment.
Insufficient blocking of nonspecific sites	Increase the concentration of protein in the blocking buffer.
	Optimize blocking time and/or temperature. Block for at least 1 hour at room temperature (RT) or overnight at 4°C.
	Adding Tween 20 detergent to the blocking buffer can help minimize background. However, too much detergent can interfere with antibody binding. A final concentration of 0.05% often works well. For ease of use, choose a blocking buffer that already contains 0.05% Tween 20 detergent, such as Thermo Scientific StartingBlock T20 Blocking Buffer (TBS) or (PBS) or SuperBlock T20 Blocking Buffer (TBS) or (PBS).
	Prepare antibody dilutions in a blocking buffer that contains 0.05% Tween 20 detergent.
	Use Thermo Scientific SuperSignal Western Blot Enhancer to reduce background and enhance detection of low-abundance and weakly immunoreactive antigens.
Insufficient washing	Increase the number of washes and/or the volume of buffer used.
Insufficient washing	Add Tween 20 detergent to the wash buffer to a final concentration of 0.05%. If the concentration of Tween 20 detergent is too high, it can strip proteins off the membrane.
Membrane handled improperly	Wet and activate membrane according to manufacturer’s instructions.
	Always wear clean gloves or use forceps when handling membrane.
	Cover the membrane with liquid at all times to prevent drying.
	Use agitation during all incubations.
	Handle membrane carefully—damage to the membrane can cause nonspecific binding.
Contamination of equipment or materials	Prepare fresh buffers and filter them before use.
Contamination of equipment or materials	Use only clean and contaminant-free electrophoresis equipment, blotting equipment, and incubation trays.
Signal from chemiluminescent substrate too strong	Reduce the length of time the blot is exposed to film.
	Reduce the concentration of the substrate.
	Shorten incubation time of membrane with substrate.
	Completely remove substrate after incubation period.
	Decrease the concentration of antibodies, particularly HRP- and AP-conjugated antibodies.

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Weak or no signal

Possible cause	Solutions
Incomplete or inefficient transfer	After transfer, stain the gel with a total protein stain to determine transfer efficiency.
	After transfer, assess transfer efficiency by staining the membrane with the Thermo Scientific Pierce Reversible Protein Stain Kit (PVDF) or (Nitrocellulose membranes).
	Ensure sufficient contact between the gel and membrane during transfer by using a gel roller across the transfer stack.
	Ensure that the stack is placed in the transfer apparatus in the proper orientation such that proteins will migrate onto the membrane.
	Wet and activate the membrane according to the manufacturer’s instructions.
	Use a positive control, such as prestained molecular weight markers, to assess transfer efficiency.
	Use molecular weight markers compatible with a western- imaging substrate, such as the Invitrogen iBright Prestained Protein Ladder or Invitrogen MagicMark XP Western Protein Standard, as a positive control.
	Increase transfer time and/or voltage.
	Make sure sample preparation conditions have not destroyed the antigenicity of the sample. (Some proteins cannot be run under reducing conditions.)
Insufficient binding to membrane	For low molecular weight (MW) antigens, add 20% methanol to the transfer buffer to help binding and prevent proteins from passing through membrane.
	Reduce transfer time. Low MW antigens may pass through membrane.
	For high MW antigens, add 0.01–0.05% SDS to transfer buffer to pull proteins from the gel onto membrane.
	Change membrane type (NC vs. PVDF).
	Change to membrane with smaller pore size.
Antibody concentration too low	Increase antibody concentrations. Antibody may have poor affinity for the target protein.
Antibody concentration too low	Antibody may have lost activity. Perform a dot blot to determine activity.
Insufficient antigen present	Load more protein onto the gel.
Antigen masked by blocking buffer	Decrease concentration of protein in blocking buffer.
Antigen masked by blocking buffer	Try a different blocking buffer. Use our blocking buffer selection guide to find the most compatible blocking buffer for your experiment.
Buffer contains sodium azide	Sodium azide inhibits HRP. Do not use it with HRP-conjugated antibodies.
Signal from chemiluminescent substrate too weak	Increase incubation time of membrane with substrate.
	Increase film exposure time.
	Ensure that the substrate is not expired.
	When you have minimal protein, use Thermo Scientific SuperSignal West Femto Maximum Sensitivity Substrate to maximize your western blot signals.
Membrane has been stripped and reprobed	Avoid repeated stripping of the same membrane.
Membrane has been stripped and reprobed	Shorten incubation time in stripping buffer to prevent loss of antigen.
Digestion of antigen on membrane	Blocking substance may have proteolytic activity (e.g., gelatin).
Protein degradation from prolonged blot storage	Prepare new blot.

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Fluorescent western blotting troubleshooting

Fluorescent western blot troubleshooting nonspecific bands

Nonspecific or diffuse bands

Possible cause	Solutions
Poor antibody specificity for the target of interest	Evaluate additional primary antibodies.
Poor antibody specificity for the target of interest	Use only primary antibodies validated* for western blots.
Poor sample integrity	Sample degradation due to overheating or protease activity results in target breakdown and low target recognition by the antibody. For example, do not boil SDS-PAGE samples in SDS sample buffer, but rather heat them at 70°C for 10 minutes to avoid proteolysis.
Antibody cross-reactivity in multiplex detection	Choose primary antibodies raised in distantly related host species.
	Use highly cross-adsorbed secondary antibodies.
	Reduce the amount of the secondary antibody used, to remain within the optimal performance range.
Fluorescent bleed-through from another channel when multiplexing (appearance of an unexpected band)	Avoid spectrally close conjugates, especially when the signal is very strong.
	Ensure that your fluorescent dyes can be distinctly detected on your imager.
	Use the autoexposure feature on the instrument to determine the optimal exposure time for each channel.

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Weak or no signal

Possible cause	Solutions
Poor antibody specificity for the target of interest	Increase primary antibody concentration.
	Ensure primary antibody has a good titer and is specific for the antigen to be detected.
	For a low-abundance target in a cell or tissue lysate, increase the amount of primary antibody or the amount of sample loaded on the gel.
	Extend the incubation time to overnight at 4°C, or 3–6 hours at room temperature.
	Try using an antibody enhancer.
Lost activity of antibody	Ensure the antibody was stored appropriately.
	Check the expiration date of the antibody.
	Avoid multiple uses of prediluted antibodies.
Imaging exposure time is too short	Increase exposure time.
Imaging exposure time is too short	Utilize the Smart Exposure feature to obtain an optimal image on the iBright FL1000 system.
Incorrect instrument settings	Ensure the correct excitation and emission ranges are selected for the intended fluorophore.
Use of detergent	Too much detergent or the nature of the detergent can result in washing away the signal—decrease or eliminate detergent.
Blocking buffer blocks antigen	Some blocking solutions can mask the blot and reduce the availability of the antigen to the antibody, especially if the blocking step is >1 hour.
	Dilute the primary antibody in wash buffer.
	Evaluate another blocking buffer.
Quantity of sample loaded on the gel	Too much lysate can overcrowd your specific target and reduce the antibody sensitivity.
	Too little lysate leads to insufficient availability of the target of interest.
	Perform serial dilutions of the lysate or sample to determine the optimal amount of protein to load.
Poor transfer of protein, or loss of the protein after transfer	Check transfer conditions to confirm protein transfer.
	Reoptimization may be required when probing for a new protein.

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Fluorescent western blot troubleshooting, high background

Background issues (high, uneven, or speckled)

Possible cause	Solutions
High background due to membrane contamination	Handle the membrane using clean forceps and clean incubation trays or dishes.
	Determine the best blocking buffer for your application— primary antibodies will react differently in different blocking buffers. Blocking buffers like normal animal sera or milk may result in cross-reactivity.
	Limit the use of detergents during blocking steps, as common detergents can auto-fluoresce and actually increase nonspecific background. After blocking, detergents can be used.
Artifacts from overloading the protein marker or ladder	Load less of the molecular weight marker onto the gel.
Nonoptimal wash or diluent solutions	Use a wash buffer with 0.1–0.2% Tween 20 detergent.
	Prepare the secondary antibody dilution with 0.05% Tween 20 detergent.
	Increase the number or duration of wash steps.
High background from an excess of secondary antibody	Optimize the secondary antibody dilution depending on the dye being used, following the vendor-recommended dilution and adapting accordingly.
Blotchy or uneven background due to the membrane drying out	Ensure good coverage of the whole blot during all incubation steps.
Blotchy or uneven background due to the membrane drying out	Ensure consistent agitation during every incubation step.
Incorrect choice of membrane	The nature of the membrane can affect the background; for example, PVDF membranes can autofluoresce and cause high background, so use low-fluorescence PVDF membranes.
Speckles and fingerprints on the membrane	Use clean forceps to handle the membrane and avoid directly touching the membrane; particulates and contaminants from unclean tools may fluoresce.
	Use clean incubation trays or dishes—rinsing with methanol followed by water will help dissolve residual dried dyes from previous uses.
	Clean transfer devices and dusty consumables if using a wet transfer method, as they can introduce speckles.
	Clean the imager surface with ethanol to remove dust, lint, and residue before capturing the image.