Western Blot Doctor™ — Signal Strength Problems

The Western Blot Doctor is a self-help guide that enables you to troubleshoot your western blotting problems. In this section, you can find solutions to problems with blot signal detection.

Other sections in the Western Blot Doctor:

Problems and Solutions

Click on the thumbnail that is most representative of your own blot to discover the probable causes and find specific solutions to the problem.

An unevenly transferred blot – Western Blot Doctor - Protein Transfer Issues

Uneven signal across blot
A blot with signal saturation – Western Blot Doctor - Signal Saturation Issues

Saturated band signal
An under-transferred blot – Western Blot Doctor - Protein Transfer Issues

Faint high-MW bands
A blot with weak-signal – Western Blot Doctor - Weak-Signal Detection

Faint bands, weak or no signal
 

Problem: Uneven signal across blot

Some sections of the blot appear to have less protein on them.

 An unevenly transferred blot – Western Blot Doctor - Protein Transfer Issues
Possible causes: Solutions:

Membrane not uniformly wet before transfer
  • Ensure that membranes are uniformly wet before transfer
  • When using a hydrophobic PVDF membrane, it must be completely soaked in methanol prior to equilibration in aqueous transfer buffer. A completely wet PVDF membrane has a gray, translucent appearance

Membrane allowed to dry during handling
  • High-intensity or rapid transfer methods generate heat that may cause the membrane to dry while it is handled. Ensure that warm membranes are not allowed to dry after transfer by shortening handling time

Buffer tank not filled to correct level
  • Completely fill transfer tank with buffer. Transfer tank must contain sufficient buffer to entirely cover blot area

Not enough solution used during incubation and/or washing
  • Make sure the membrane is fully immersed and agitated throughout incubations

Uneven agitation during incubation
  • Use a shaker for all incubations
 

Problem: Saturated band signal

Bands are solid black. If using a digital imager, you see regions highlighted as saturated.

 A blot with signal saturation – Western Blot Doctor - Signal Saturation Issues
Possible causes: Solutions:

Sample overloading
  • Decrease total protein loaded on gel
  • To optimize sample loading, see Determining the Appropriate Sample Load for Western Blots Protocol

Too much antibody
  • Decrease concentration of primary and/or secondary antibodies
  • Optimize your primary and/or secondary antibody concentrations using a checkerboard screening protocol

Exposure too long
  • Use a shorter exposure
  • Use multi-acquisition feature on data acquisition software

Rapid substrate consumption
  • Reduce incubation time with detection substrate
  • Use a less sensitive detection substrate
 

Problem: Faint high-MW bands

High-molecular-weight bands look fainter than other bands on the blot
(see also Protein transfer or binding issues below).

 An under-transferred blot – Western Blot Doctor - Protein Transfer Issues
Possible causes: Solutions:

Transfer conditions not optimized:

  • Transfer time too short
  • Transfer power inadequate
  • Transfer buffer formulation

Improper gel selection
  • For best transfer of large proteins, use a low-percentage gel, e.g., a 7.5% or 4–15% gradient gel, and add 0.05% SDS to the transfer buffer
  • Detect any residual protein remaining in the gel by staining and imaging the gel post-transfer, or use stain-free technology to image total protein on both the gel and the blot before immunodetection. Bio-Rad’s V3 Western Workflow™ enables you to visualize, verify, and validate protein separation and transfer at every step of the electrophoresis and blotting procedure
 

Problem: Faint bands, weak or no signal

(see also Protein Band Appearance > No Bands)

All bands appear very faint, or you see no signal with chemiluminescence or fluorescence detection.

 A blot with weak-signal – Western Blot Doctor - Weak-Signal Detection
Protein transfer or binding issues
For more hints on how to improve transfer and binding, see also Troubleshooting: Transfer in the Protein Blotting Guide
Possible causes: Solutions:

Insufficient sample loaded on the gel

Low transfer efficiency
(see below for more details on transfer power conditions, gel electrophoresis, and buffer preparation; see also Western Blotting > Transfer Conditions)
  • Confirm transfer with Ponceau S staining
  • Confirm transfer with Bio-Rad’s Stain-Free Imaging Technology
  • Optimize transfer conditions for target protein size
  • Lower gel acrylamide percentage
  • Increase transfer time
  • Optimize transfer buffers for methanol and SDS concentrations

Power conditions inadequate or transfer time too short
  • Increase the transfer time (thicker gels require longer transfer times)
  • Check the current at the beginning of the run; it may be too low for a particular voltage setting, indicating incorrect buffer composition. See the power guidelines for specific applications in Table 4.1 of the Protein Blotting Guide (Bulletin 2895)
  • Use high-intensity blotting devices, e.g., Semi-Dry and Rapid Blotting Systems such as the Trans-Blot® SD or Trans-Blot® Turbo™ Systems
  • Use a power supply with a high current limit such as the PowerPac™ HC Power Supply. If an incorrect power supply is used, it may not reach the set voltage if the current of the power supply is at its maximum limit

Power conditions too high or transfer time too long

  • Low-MW proteins may be transferred completely through the membrane
  • Proteins <15 kDa may show decreased binding to 0.45 µm membranes
  • Reduce transfer time
  • Decrease the voltage, especially if using a high-intensity transfer device, e.g., Semi-Dry and Rapid Blotting Systems such as the Trans-Blot SD or Trans-Blot Turbo Systems
  • Place an additional membrane in the gel sandwich to detect any low-MW proteins that are transferred through the membrane
  • Use PVDF or 0.2 µm nitrocellulose (smaller pore size)

Power supply circuit inoperative or an inappropriate power supply used
  • Check the output capacity of the power supply to ensure the voltage and current output match the needs of the blotting instrument. If it does not match your needs, get a power supply with a higher current capacity such as the PowerPac™ HC Power Supply
  • Check the fuse

Protein transferred away from the membrane
  • Check that the gel/membrane sandwich has been assembled in the correct order
  • Check the assembly of the transfer cell, and ensure that the cassette is inserted into the tank in the correct orientation
  • Check the polarity of the connections to the power supply

Incorrect charge-to-mass ratio (native transfers)
  • Use a more basic or acidic transfer buffer to increase protein mobility. A protein near its isoelectric point (pI) will transfer poorly (buffer pH should be 2 pH units higher or lower than the pI of the protein of interest for optimal transfer efficiency)

Protein precipitated in the gel due to

  • High protein concentration
  • Low solubility
  • Partial folding/aggregation
  • Use SDS in the transfer buffer. SDS can increase transfer efficiency but it can also reduce binding efficiency to nitrocellulose and affect reactivity of some proteins with antibodies
  • Reduce or eliminate the alcohol in the transfer buffer

Gel percentage too high, decreasing transfer efficiency
  • Reduce %T (total monomer) or %C (crosslinker) to increase gel pore size and increase transfer efficiency. Using 5% C (with bis-acrylamide as the crosslinker) produces the smallest pore size
  • For example, generally protein gels are around 29:1 acrylamide-to-bis-acrylamide. If you are using 19:1, you should try 29:1. If you are examining a large complex and using 29:1, you may want to try 37.5:1 or try higher ratios. A higher acrylamide to bis ratio results in a gel with larger pores

Transfer buffer incorrect or prepared incorrectly
  • Prepare fresh transfer buffer (for best results, never reuse transfer buffer)

Methanol in the transfer buffer inhibiting elution
  • Reduce the amount of methanol. This may improve transfer efficiency of proteins from the gel but it may also decrease binding to nitrocellulose membranes; 20% methanol is generally optimal for protein binding
Detection issues

For more tips on resolving immunodetection problems, see also Troubleshooting: Detection in the Protein Blotting Guide
Possible causes: Solutions:

Primary or secondary antibody concentrations too low
  • Repeat using higher concentration of antibody
  • Optimize antibody concentration with dot blots
  • Checkerboard screening protocols

Low antigen-antibody binding affinity
  • Reduce the number of wash steps to a minimum
  • Reduce wash stringency
  • Increase the antibody concentration 2–4 fold higher that the recommended starting dilution
  • Checkerboard screening protocols

Blot has been stripped and reprobed
  • Redo blot because antigen may have been stripped off or damaged by stripping process

Film exposure time (ECL detection) too short

Nonfat dry milk may mask some antigens
  • Decrease percentage (w/v) of milk in the blocking and antibody solutions
  • Try alternate blocking solutions (e.g., albumin, gelatin, or BSA)

Insufficient sample loading
  • Increase the amount of protein applied
  • Concentrate the sample prior to loading

Detection system not working or not sensitive enough
  • Use a more sensitive assay system
    • If using colorimetric detection try an enhanced chemiluminescent substrate (ECL) such as Bio-Rad’s Clarity™ ECL Substrate
    • If using alkaline phosphatase (AP) conjugate, try a horseradish peroxidase (HRP) conjugate instead (see our selection of HRP and AP Conjugates)
    • If using a general ECL substrate, try a high-sensitivity ECL substrate
  • Include proper positive and negative control antigen lanes to test for system sensitivity; consult your detection kit manual

Proteins washed from the membrane during assays
  • Reduce the number of washes or reduce the stringency of washing conditions during subsequent assay steps
    • Reduce the time of each wash step
    • Reduce washing temperature
    • Reduce the concentration of detergent (usually Tween 20) in the wash buffer, or remove it entirely from the wash buffer
    • Reduce the salt concentration in the buffer

Insufficient antigen binding to the membrane
  • Stain the blot after transfer or use prestained standards to assess transfer efficiency. Alternatively, use stain-free technology to assess sample being on the blot. 
  • See the previous section for suggestions on improving transfer-related problems

Antigen denaturation during electrophoresis or transfer
  • Antibodies, especially monoclonals, may not recognize denatured antigens. If this is the case, electrophorese and transfer proteins under native conditions, e.g., blue native PAGE. Use a cooling coil and a refrigerated recirculating bath to transfer heat-sensitive proteins

Epitope blocked by total protein stain
  • Some total protein stains (such as amido black and colloidal gold) interfere with antibody recognition of the antigen. Try a different stain (e.g., Ponceau S) or Bio-Rad’s Stain-Free Technology

Primary or secondary antibody inactive or nonsaturating
  • Store the reagents at recommended conditions in small aliquots to avoid repeated freeze-thaw cycles, bacterial contaminants, and heat inactivation
  • Detergents may affect the binding of some antibodies. Eliminate them from the assay, except for the wash after blocking
  • If the antibody titer is too low, optimize the concentrations using a dot-blot experiment (see Protein Blotting Guide: Microfiltration)
  • Optimize antibody concentrations using a checkerboard protocol.
  • Increase the antibody incubation times
  • Test the activity of the first antibody solution. Use an ELISA, RID, Ouchterlony immunodiffusion, or precipitation test to determine the reactivity of the antibody with the antigen. If possible, repeat the assay procedure with a more concentrated primary antibody solution

Chemiluminescence substrate or color development reagent inactive
  • Test the activity of the color development solution by combining 1.0 ml of the color development solution with 10 µl of full-strength secondary antibody conjugate. The color reaction should occur immediately. If color fails to develop within a few minutes, the color development solution is inactive. Prepare a fresh working solution and repeat the color development assay

Enzyme conjugate inactive or nonsaturating
  • Test the activity of the conjugate solution by combining 1.0 ml of the color development solution tested above and 1.0 ml of the 1:3,000 dilution conjugate solution. A light-blue tinge should develop within 15 min. If color fails to develop within 25 min, the conjugate solution is suspect. Repeat the procedure with a freshly prepared dilution of conjugate
  • Store the reagents at recommended conditions. Avoid repeated freeze-thaw cycles, bacterial contaminants, and heat inactivation
  • Sodium azide inactivates horseradish peroxidase. Use a different biocide such as gentamicin sulfate instead
  • Undistilled water may cause inactivation of the enzyme. Use only distilled, deionized water
  • If the conjugate concentration is too low, optimize using a dot-blot experiment (see Bio-Dot® and Bio-Dot SF Microfiltration Apparatus)
  • Optimize antibody concentrations using a checkerboard protocol
 
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