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Troubleshooting Tips

Troubleshooting tips for western blotting

Troubleshooting tips for western blotting common problems: 
    1.High background
    2.Low or no signal
    3.Non-specific bands
    4.Wrong band location
    5.Invisible dots on the bands
    6.Incomplete bands
    7.Smile effect of the bands
    8.White bands on a black blot 
    9.Black dots on the blot
    10.Marker lane is black


1.High Background

Causes Solutions
Membrane fouling Use clean tweezer and operate with gloves to prevent membrane fouling.
The membrane has dried out Incubate in sufficient reaction solution to prevent the membrane from drying out.
Blocking insufficient Increase the blocking incubation time
Inappropriate blocking buffer Switch different blocking buffer
Buffer solution has been contaminated Use new buffer solution
Incomplete washing Increase washing time and washing buffer’s volume
The antibody concentration may be too high Decrease the concentration of primary antibody or secondary antibody
Cross-reaction between antibody and blocking agent Choose blocking solution without cross-reaction. Add Tween-20 to the washing buffer to reduce cross reaction.


High background:          Improved:


 2.   Low or no signal


Causes solutions
Insufficient antigen Increase amount of loading samples
Protein degradation  Re-prepare samples
 The antigen is blocked by blocking buffer  Optimize blocking solution, decrease blocking time or  decrease the concentration of proteins in the blocking solution.
 No or low level of target protein in samples  Run a positive control. If the level of target protein in samples is low, try to increase amount of loading sample.
 Wrong choice of membrane  Choose suitable pore size membrane. Use 0.45um size membrane for proteins larger than 22KD. Use 0.2 µm size membrane for proteins smaller than 22 KD.
 Poor transfer of protein to membrane  Make sure there are no air bubbles between the gel and membrane during transfer. Always ensure assembling electrode correctly. Control transfer temperature and optimize transfer electricity and time.
 Methanol concentration may be too high.  Too high concentration of methanol may result in the separation of protein and SDS and thus cause protein precipitation  in the gel. At the same time, it may cause shrinking or hardening of the gel to inhibit transferring of high molecular weight proteins. As a result, choose suitable methanol concentration according to different molecular weight.
 Excessive washing of membrane  Reduce washing time and washing times
 Over blocking  Lower the concentration of your blocking solution and shorten blocking time. Change blocking solutions.
 The primary antibody is inactive  Use effective antibody in expiration, avoid freezing- thawing repeatedly, and use fresh solution.
 Insufficient reaction of antibody to membrane  Increase the concentration of the antibody and the incubation time.
 The reagents are not compatible with each other  Primary antibody and species, primary antibody and secondary antibody, or enzyme and substrate are not compatible. Setting loading control can validate the secondary detecting system.
 HRP Inhibited  Avoid sodium azide in all solutions and containers
 Enzyme or substrate is inactive.  Directly mix enzyme and substrate. If no color, the enzyme doesn’t work. Choose active conjugated reagent. Use fresh substrate.
 Exposure time is too short  Increase the exposure time 


 Low or no signal     Improved


3. Non-specific bands

Causes Solutions
Amount of loading samples is too large Decrease amount of loading samples
Protein degradation Use fresh samples and use protein inhibitor
Cells were cultured too many passages to result in protein variation Use primary cells or less passaging cells to run a control
Antibody Concentration is  too high Decrease the concentration of the primary or secondary antibody
Cross-reaction Choose monoclonal antibody or affinity purified antibody to ensure antibody specificity
Existence of the different protein  isoforms Some proteins derived from the same gene have different isoforms. The size of every isoform protein is different.
Non-specific signal caused by the secondary antibody Run a secondary antibody control or choose other secondary antibody
Substrate is too sensitive Use suitable substrate
Exposure time is too long Reduce the exposure time


Non-specific bands:   Improved:


4.Wrong band location

Causes Solutions
Existence of dimer or polymer Increase a process or intensity of protein denaturation
Modification of proteins Modification of proteins, such as glycosylation or phosphorylation, can result in an increase of molecular weight of protein.


5.Invisible dots on the bands

Causes Solutions
Air bubbles were trapped in the gap of gel and membrane during transferring. Remove bubbles in the gap of gel and membrane when preparing for transferring.


Invisible dots on the bands:   Improved:


6.Incomplete bands 

Causes  Solutions
Substrate is not well-distributed during incubation Even the substrate during incubation


Incomplete bands: Improved:


7.Smile effect of the bands


Causes Solutions
Migration was too fast during electrophoresis Reduce the voltage to slow down the migration
Migration was too hot Run the gel in the cold room or in ice


Smile effect of the bands:  Improved:


8.White bands on a black blot

Causes Solutions
HRP concentration is too high Decrease the secondary antibody concentration


White bands on a black blot:  Improved:


9.Black dots on the blot

 Causes  Solutions
 Air bubbles were trapped against the membrane during transferring or the antibody is not well distributed during incubation  Try to remove bubbles. Keep shaking when incubating the antibody.
 The blocking agent was not well dissolved.  Filter the blocking agent.
 The antibody reacts with the blocking solution.  Choose suitable blocking solution
 There are aggregates in the HRP conjugated secondary antibody.  Filter the secondary antibody agent and remove the aggregates.


Black dots on the blot:      Improved: 


10.Marker lane is black

Causes Solutions
The antibody reacts with the MW marker Add a blank lane between the marker and the adjacent sample lane