Posted by M.Popov on 12th Nov 2020
Troubleshooting issues with Western Blot background with St John's Laboratory
Troubleshooting issues with Western Blot background with St John's Laboratory
Introduction:
In this article we discuss and provide solutions for common Western Blot background problems, but if you have any other issues with this analytical technique, please consider using our other articles about issues with signal strength, protein band size and pattern and band appearance.
During Western Blotting, several issues may occur with the background:
- White regions on the background
- High background across the blot
- Patchy background
- Uneven spots on blot or background
- Shadow transferred on the blot (tank blotters)
White regions on the background
White areas on WB background can be caused by air bubbles or dry areas of membrane restricting proteins from binding. Both can be easily prevented with improving your technique and additional care.
| Causes: | Solutions: |
| Faulty assembly of transfer sandwich, air bubbles are present. | -Carefully remove all air bubbles present between the membrane and the gel prior to transferring the protein, either a glass rod or a roller can be used. -Using a rapid transfer pack greatly helps preventing improper sandwich assembly. -If you use a tank blotter, make sure that the sponges(foam pads) can create a proper assembly and if they can no longer create a good seal, the sponges should be replaced with new ones. -Checking for residual protein or protein unable to transfer, remaining on the gel, using a total protein stain is advisable after transfer is complete. -In order to be sure that the transfer is successful, staining the membrane with Ponceau S after blotting is advisable. -You can visualize total protein on blots or gels using stain-free gels. -Running a diagnostic test like checking transfer quality via imaging proteins on the gel and the blot is advisable. If you want to use the blot in downstreams steps, confirm that the stain can be removed or its compatibility with antibody antibody detection. Consider that collodial gold and coomassie are not compatible with downstream steps. |
| Buffer is overheated | -Tank blotting with high-intensity transfers can lead to heating the buffer and formation of bubbles: -Ensure that the buffer is cool by reducing the current and increasing transfer time to compensate; -You can do the transfer in tank placed on an ice bath or temperature-regulated room (4C); -Chilling the buffers, using cooling coil or "blue ice" insert in the cell can help with temperature regulation. |
| Membrane is dry or not hydrated properly | -White regions on the nitrocellulose membrane show dry regions where protein is unable to bind. If sheet is not immediately immersed in transfer buffer in order to wet it, distilled water should be heated up just below 100C and the sheet should be left to hydrate inside the water. After the sheet is completely hydrated, it is left in transfer buffer until it is ready to use. -White regions on PVDF membrane show areas where the membrane was either incorrectly wetted or had the time to dry out. The easiest way to counter it is to prewet the membrane in methanol before equilibration in aqueous transfer buffer and once it is wet, do not let it dry out. |
High background across the blot
If high background is seen across the blot, several things may be the reason. Following the protocol and careful handling ensures that the experiment runs smoothly, but several trials may be needed to resolve this problem.
| Causes: | Solutions: |
| Incomplete membrane blocking | -Concentration of blocker can be increased(3-5% BSA, casein or nonfat dry milk). -Increase of duration of the blocking step, for example 8 hours at 4C instead of an hour at room temperature. -Increase of temperature at which blocking is performed. -Different blocking agents may help(gelatin, BSA, albumin, casein, nonfat dry milk) |
| Blocking reagent has been contaminated | -Using pure protein such as BSA or casein as a blocker can help. -Reusing blocking buffers is not advisable. |
| Incubation with substrate for too long | -Shorter incubation time with detection substrate. -If colorimetric reagent is used, removing the blot from the substrate when the signal-to-noise level is appropriate and immersing in deionized water is advisable. |
| Excess use of antibody | -Optimizing the concentrations of primary or secondary antibodies is the best solution. -Using a dot-blotting trial to optimize antibody concentrations. -Reduction or optimization of incubation times. |
| Incubation tray has been contaminated | -Fully clean the incubation trays between experiments. -Using disposable trays can help with contamination. |
| Tank blotter specific problems: -Excessive protein loading -Excess SDS in transfer buffer | -Smaller amount of protein on gel. -Optimize sample loading and reduce the concentration of SDS in transfer buffer. -A second membrane may help to bind excess protein. |
| Antibody binding to proteins in blocking buffer | -Use other blocking reagents/BSA, casein, gelatin, albumin or nonfat dry milk/ |
| Inappropriate washing between incubation steps | -Use more wash buffer -Increase the length or number of washing steps/minimum 5x5 min/ |
| Too long exposure | -Shorter exposure time -Multi-acquisition feature on data acquisition software might help -For film, waiting between 5 to 10 minutes and re-exposing blot to film afterwards or reducing the exposure and development time of the film might help |
| Membrane has dried out during blotting procedure incubation steps | -Try to wet the membrane thoroughly when you begin the procedure -Keep the membrane submerged in incubation or wash buffers in all the steps |
| Antibody activity loss due to improper storage | -Storing an antibody for a prolonged period should be at -80C - Use a fresh aliquot that has been stored at -20C or below and avoid freeze-thaw cycles -Make aliquots and only thaw one at a time as needed for experiments |
| Too high incubation temperature | -Try lower temperature such as 4C, but increase incubation time |
| PVDF has higher background than nitrocellulose | -Use nitrocellulose instead |
| Insufficient concentration of detergent in buffers | -Use a stronger detergent such as NP-40 -Use TBS with 0.05-0.1% Tween 20 |
| Contaminated buffers | -Fresh buffers are advisable -All buffers should be filtered at 0.2 µm filter before use |
Patchy background
Patchy or blotchy background can be caused by insufficient washing or the membrane drying out. By making sure that the blot is immersed at all times and preventing contamination from laboratory sources this problem is easily eliminated.
| Causes: | Solutions: |
| Agitation during incubations is uneven | -Using a shaker throughout all the incubation steps is advisable. |
| Patches of the membrane have dried out | -Confirm that the membrane is completely wet before starting the procedure. -Make sure that the blot does not dry out throughout the procedure and that the blot is always submerged. |
| Blot contamination | -Using clean gloves is essential and handling blots with clean forceps is highly advisable. -Do not rest the blot on surfaces, which can contaminate it. |
| Some areas of the blot have not been properly washed or accessed by the incubation buffer | -Utilising a shaker during the wash and incubations steps is advisable. -Higher washing buffer volume helps with reaching all parts of the blot. -Stacking blots in a single incubation chamber is not advisable unless good flow of buffer exists between the blots and that they can move freely during the steps of incubation and agitation. |
Uneven spots on blot or background
Outside material adhering to the membrane, not properly cleaned cassette or dirty scanner surfaces and antibody aggregation can cause uneven spots. Improving handling procedures prevents these problems.
| Causes: | Solutions: |
| Secondary antibody is aggregated | -Spinning the secondary antibody or filtering it will remove aggregates. |
| Cluster of blocking reagent is stuck to the membrane or the antibodies are binding to the blocking reagent | -Completely dissolve the blocking agent in the buffer before using. -Making fresh buffer if the blocking is made from dry reagents is advisable. -Consider observing premade blocking buffers forany precipitates before using. -Adding 0.05-0.1% Tween to blocking buffer is recommended. -Pouring the blocking buffer through 0.2 µm filter before using it is advisable. -Prior to incubation with antibodies, consider washing the blot with washing buffer. -A different reagent can be used, for example BSA, gelatin, nonfat dry milk, casein or albumin. |
| Gel particles are stuck to the memrbane | -All of the residual acrylamide gel traces should be removed from surface of the membrane after the transfer prior to processing with downstream steps. |
| Contaminated cassette and scanner surfaces | -Make sure that both the cassette and scanner are completely clean before proceeding. |
| Buffer is contaminated | -Using fresh buffer is preferable. -Try making a new batch of buffer and repeat the procedure. -Passing the buffers used for blocking and washing through a 0.2µm filters removes the particles from them. - Blot drying at the image acquisition step and particulate contamination can be prevented by wrapping the blots in either heat-sealed bags or plstic wraps. |
Shadow transferred on the blot (tank blotters)
If extended transfer is used, the pattern of the transfer cassette plastic or the holes can be seen on the blot. Often the reason for this problem is that the foam pads in the sandwich are too thin or a protein contamination is present.
| Causes: | Solutions: |
| Too thin or contaminated foam pads | -Cleaning the pads if they are contaminated and replacing them with new ones if they are deformed is advisable. |
| Too much protein is loaded on the gel and in a tank blotter any excess amount of protein is able to pass through and recirculate in the buffer. | -Optimise the protein on the gel and samle loading. -Lower the amount of SDS in the transfer buffer. -A second membrane helps bind any excess protein. |
| Contamination of the transfer buffer | -Using fresh transfer buffer and making a new batch in order to repeat the blot is advisable. - Consider passing the blocking and washing buffers through 0.2 µm filter in order to remove contaminants. |