Hello Ritika,

You may switch to a different vendor for your primary antibody. The present primary antibody may contain impurities that could cause non-specific binding. It might recognize similar epitopes on other proteins, leading to multiple bands. Additionally, dilute the secondary antibody further from 1:10000 to 1:20000.

You may want to refer to a similar thread on ResearchGate for other reasons of obtaining multiple non-specific bands on Western Blot.

https://www.researchgate.net/post/Reason_for_non-specific_bands_in_Western_blotting

Best,

There are several reasons for nonspecific bands in Western blotting, including:

Antibody:

Cross-reactivity: The antibody may bind to proteins other than the target, especially if the proteins have similar amino acid sequences.

High antibody concentration: Using high antibody concentrations can increase the likelihood of the antibody binding to non-target proteins.

Polyclonal antibodies: Polyclonal antibodies are a mixture of antibodies that recognize different epitopes on the target protein. This mixture can contain antibodies that also bind to non-target proteins.

Sample preparation:

Protein degradation: Proteins can degrade during sample preparation, creating additional bands.

Protein Modifications: Proteins can undergo modifications such as phosphorylation or glycosylation, which can change their molecular weight and create additional bands.

Protein Aggregation: Proteins can aggregate together and create higher molecular weight bands.

Western Blot Method:

Insufficient Transfer: Insufficient transfer of proteins from the gel to the membrane can result in blurred or faint bands.

Insufficient Washing: Insufficient washing of the membrane can result in non-specific antibody binding.

Insufficient Blocking Conditions: Insufficient blocking of the membrane can result in non-specific antibody binding.