The usually encountered problems of western blot and the solution in accordance.

The western blot (also called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology, and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detecting the proteins, this technique is also utilized to visualize, distinguish, and quantify the different proteins in a complicated protein combination. The western blot method is composed of a gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide, followed by an electrophoretic transfer onto a membrane (mostly PVDF or nitrocellulose) and an immunostaining procedure to visualize a certain protein on the blot membrane. (Source: Wikipedia https://en.wikipedia.org/wiki/Western_blot).


As the procedure is complicated, the results are also impacted by diverse factors. This article will summarize the usually encountered problems and the solution in accordance.


1. No bands or no target bands on the membrane


Problem Possible reasons Suggestion
Operation Problem Over-rinsing The rinsing time should not be too long, and the detergent should not be too strong or too much. Try to reduce the number of rinsing times, rinsing time and the temperature.
The protein has not been transferred to the membrane After the transfer, determine the ransfer efficiency with the total protein stain; during the transfer process, the gels should be in complete contact with the membrane; the transfer “sandwich” is arranged correctly; wet the membrane according to the instructions of the membrane manufacturer; transfer parts not overheating during the electro-blotting process; stain the membrane with Ponceau and combine with staining gel (Coomassie Brilliant Blue) to determine whether the band is transferred to the membrane or over-transfer; adjust appropriately the transfer time and current .
The protein has not fully been transferred to the membrane Low-molecular-weight antigens may pass through the transfer membrane. We can use a small pore size membrane.
Over-blocking Reduce the blocking time, try different blocking fluid.
Substrate incubation time is too short Extend the substrate incubation time.
Antibodies Primary antibody and secondary antibody do not match Choose the type of secondary antibody which matches the primary antibody. The secondary antibody must be of the same species as the primary antibody host. The effectiveness of the secondary detection system can be verified by setting internal parameters.
Primary antibody failure Use the antibody within the validity period and store it in aliquots to avoid repeated freezing and thawing. The working fluid should be freshly prepared before use.
Antibodies combined to the target protein are insufficient  Reduce the dilution of the primary antibody; use a high-effective primary antibody; extend the antibody incubation time; fully immerse the membrane in the fluid.
Low concentration of antibodies Increase the concentration of antibodies
High concentration of antibodies Using too much primary or secondary antibody may cause rapid depletion of the substrate and showing a weak signal
Antigen The primary antibody does not recognize the target protein Compare the antigen sequence and protein sequence with reference to the database, select the appropriate primary antibody
Insufficient or degraded antigen The sample amount in each lane should not be less than 20~30 ug, use protease inhibitor and set a positive control.
Blocking substrate may contain proteolytic enzyme activity
Blocking fluid The blocking fluid cross-reacts with the primary or secondary antibody Use appropriate blocking fluid
Buffer contains Sodium azide Sodium azide is an inhibitor of HRP. Sodium azide cannot be used as a preservative in buffers
ECL luminescent fluid is inactivated or not sensitive enough Use fresh and sensitive ECL
Insufficient exposure time Extend exposure time


2. Target bands emit weak signals


Problem Possible reasons Suggestion
Operation The protein has not fully been transferred to the membrane Standardize the operation of membrane transfer; optimize the conditions of electrotransfer; two membranes should be superimposed together to prevent excessive transfer, or use a small pore membrane.
Over-rinsing The rinsing time should not be too long, and the detergent should not be too strong or too much. Try to reduce the number of rinsing times, rinsing time and the temperature.
Over-blocking Reduce the blocking time, try different blocking fluid.
Insufficient exposure time Extend exposure time
Insufficient antibody staining Extend incubation time or increase antibody concentration
Antibodies Low concentration of antibodies Increase concentration of antibodies
Insufficient affinity between antibody and antigen Increase concentration of antibodies
Insufficient antibody activity Use the antibody within the validity period and store it in aliquots to avoid repeated freezing and thawing. The working fluid should be freshly prepared before use.
Antigen Insufficient antigen Increase sample volume
Blocking fluid Buffer contains Sodium azide Sodium azide is an inhibitor of HRP. Sodium azide cannot be used as a preservative in buffers.


3. The background of the images is too high.

3.1 Uniform high background

Problem Possible reasons Suggestion
Operation Temperature too high during blocking，blocking is insufficient Selection and optimization of the blocking fluid; use 5% skimmed milk powder; optimize the blocking time and temperature
Membranes Prevent membranes drying; use NC membrane for experiment; use clean tweezers and wear gloves during operation
Insufficient rinsing during incubation Properly increase the time and frequency of washing the membrane; ensure that the membrane is incubated under sufficient shaking conditions and the antibody fully covers the membrane surface
Overexposure Shorten the exposure time, or use imaging systems
Antibodies High concentration of antibodies Increase the dilution of primary and secondary antibodies and optimize the dilution conditions
The blocking fluid cross-reacts with the primary or secondary antibody Replace the blocking fluid; replace the secondary antibody or reduce the concentration of the secondary antibody
Long storage time or improper storage conditions cause antibody inactivation Use fresh antibodies
Antigen Not enough detergent in the buffer Increase the concentration of Tween 20 in TBST
Blocking fluid Transfer fluid, blocking fluid are not fresh or contaminated Fluid should be freshly prepared before use.


3.2 Heterogeneous high background


Problem Possible reasons Suggestion
Operation Temperature too high during blocking，the blocking procedure is insufficient Selection and optimization of the blocking fluid;  optimize the blocking time and temperature
The membrane is not properly wetted and may have dried Use clean tweezers and wear gloves to operate; use enough liquid to keep the membrane moist; use a decolorizing shaker during incubation; avoid overlapping membranes and covering each other
Antibodies High concentration of antibodies Increase the dilution of primary and secondary antibodies and optimize the dilution conditions
Secondary antibody aggregation Filter the secondary antibody before use or replace it
Blocking fluid Aggregates in the blocking fluid Filter the blocking fluid before use or replace it
The blocking fluid cross-reacts with the primary or secondary antibody Replace the blocking fluid; replace the secondary antibody or reduce the concentration of the secondary antibody
Transfer fluid, blocking fluid are not fresh or contaminated Fluid should be freshly prepared before use.


4. A number of none-Specific bands 


Possible reasons Suggestion
Non-specific binding of antibodies to proteins Replace antibodies
Concentration of Antibody is too high Reduce the sample amount; increase the dilution of the primary antibody.
Antibody is not purified Use monoclonal or affinity chromatography purified antibodies to reduce non-specific bands
Strong transfer intensity of membrane causes  strong signal of non-specific bands Reduce the time and current of transfer
Blocking is incomplete Increase protein concentration in blocking fluid
Too many passages of cells lead to differentiation of their protein expression patterns Use the original cell line or the cell line with few passages as a reference with the current cell line


5. Shape of the bands is abnormal

5.1 Dumbbell shape


a. The gel is not uniform after solidification. We can recast the gel and ensure the quality of it.

b. The sample may contain too many impurities. We can centrifuge the sample before use to remove the excessive impurities.


5.2 Bands are white


The possible reason for this phenomenon is that the concentration of the first antibody is too high, the catalytic activity of HRP on the second antibody is too strong, and the chromogenic substrate is at the critical point, the reaction time is not long, and the surrounding substrate is catalysed completely. Therefore, the concentration of the first and second antibodies can be reduced, or the substrate can be changed.


5.3 Bands connect with each other


The possible reason for this phenomenon is that the amount of the sample loading is too high or there is a problem by gel casting. As a result, there occurs a gap between the separating and concentrating gels, or the sample is porous. This problem can be avoided by reducing the amount of sample loading or improving the quality of the gel casting.


5.4 Bands have tails


The main reason for this phenomenon is that the concentration of the protein or the first antibody is too high, or the Incubation time of the first antibody is too long. 


5.5 Band is smiling


The electrophoresis speed is too fast, and it can be slowed down by reducing the voltage. It is also possible, that the temperature of the electrophoresis is too high, which makes the gel deformed. 

Electrophoresis can be carried out in a cold room or ice bath. 


5.6 A white circel in your band


This phenomenon is probably due to the existence of bubbles between the membranes and the gels during the tranfering. 


At last, we hope, all the researchers will bring us nice images of western blot.



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