Hello Ashit Kumar Dutta

Looking at your blot it seems that you have a uniform high background that darkens the entire blot making it difficult to see specific bands. Uniform background can result from a variety of reasons.

1. Ensure your buffers are made fresh. You may want to filter them to remove dust or particulates that may be deposited on your blot and interact with your antibodies. Please note that bacterial contamination of buffers can lead to high background, especially buffers containing detergent and milk which can promote bacterial growth.

2. Make sure you select the right blocking agent that doesn’t interact with your antibody or block your epitope. Uniform high background can be observed when blocking agents interact with antibodies causing interference. For example, antibodies can bind to proteins in protein-based blocking agents and result in high uniform background. The two most common blocking agents are non-fat dry milk and BSA. It is often recommended that 5% milk be used for blocking. However, sometimes this concentration of milk can mask the protein being detected, particularly if it is not abundant. So, you begin with 1% milk. Sometimes, milk can be incompatible with certain antibodies and detection systems. In such case, 3% BSA is a second protein-based blocking agent that can be used. If neither of these agents is adequate, then protein-free or non-animal protein blocking reagents may be used. Please note that milk contains casein, a phosphoprotein that can react with phospho-specific antibodies.

3. Perform washes in large volume, change the wash solution often and agitate the membrane during the washing steps. A recommended protocol is to wash the membrane 3 times after each incubation, washing for 5 minutes per wash. The number of washes and length of wash time can be increased to improve washing efficiency. However, please note that over washing can decrease signal strength. Tween-20 (0.05%) is commonly used in wash buffers. A slightly stronger detergent, such as 0.05% NP-40 can be used to increase stringency of the wash. You should note that only highly purified detergents should be used as impurities in detergents can interfere with horse radish peroxidase detection systems. Detergent should be freshly added to wash solutions.

4. Different membrane types can affect the level of background. The age of the membrane and how membranes are handled can also impact the degree of background obtained. Nitrocellulose membranes tend to give the lowest background while PVDF membranes though have a high binding capacity give higher background. Don’t allow membranes to dry out as this will also result in high uniform background. Please note that older membranes can give high uniform background, therefore pay attention to expiration dates and use newer membranes to decrease background.

5. I need to draw your attention to the antibodies. Some initial work needs to be done (antibody optimization) to save you a lot of time down the line. Using too much antibody can increase the amount of antibody that binds non-specifically to the membrane. Start with the antibody dilution recommended by the antibody provider. I would recommend 1:5000 dilution for the primary antibody, and for the secondary antibody you may start with 1:10000 dilution and may go down up to 1:30000 dilution.

6. When over-exposed, any blot can appear as solid background. Ideally, the signal from specific bands is much stronger than any background noise, and a short exposure will pick up only the specific signal. If using film, be prepared to expose the blot multiple times to different pieces of film for increasing periods of time to find the optimal exposure time.

Hope these suggestions help!

Good Luck!

Some quick points I noticed from your blot:

the presence of black dots and other darker spots on your blot could indicate that your buffer isn’t clean. So, it’s important to use clean and fresh buffers, which are crucial for antibody-antigen interactions.

Consider using a more effective blocking buffer and ensure it’s applied for a sufficient duration. The material of the blot can vary, so if it is necessary it’s necessary to optimize the buffer conditions for each buffer. Blocking is also a key step to prevent non-specific binding.

In addition, try not to shorten or skip the washing steps since they help to remove any unbound antibodies. Also, maybe if necessary, optimize the concentration of your antibodies to get a clear signal.

Avoid overheating the blot during run. You can use an ice block or run your blot in a cold room to prevent this.

Finding the right exposure time in your detection system can also enhance the clarity of your blot.

A tip: You can check if your protein has been successfully transferred to the membrane by using Ponceau staining before proceeding with the detection.

I hope this helps!

Thank you everyone. I managed to solve the problem. I remade the antibody solution and reprobed the same blot and got much cleaner result. Leaving the new file here for reference. Again, appreciate the quick responses.

The new blot seems better, but it could still be better. Normally, I would block in 5% BSA in PBST for 1 hour and dilute my secondary antibody in 1% milk in PBST. I also mostly use nitrocellulose membranes instead of PVDF. I think you can follow the suggestions made by Malcolm Nobre

there are a few ways to improve your western blot images.

1) blocking enough (time and fresh blocking buffer;

2) further dilute your primary and secondary antibody concentrations;

3) dry your membrane to remove all ECL solution before imaging.