Hi,
I am doing Western blot for Insulin receptor Beta( from Cell signalling), on Endometrial cell line HEC 1A. The desired band size is 95kDa. I prepared 10% SDS PAGE gel, antibodies in 3% BSA in PBST, Blocking with 5% BSA. I am getting intense non specific bands in my treated proteins with IGF1( Picture attached). Need suggestion how to overcome/ trouble shoot this issues? TIA
what are the molecular weight of each of the three bands? . coukd the two small intense bands correspond to a cleavage of the higest faint band? do you use a poly or a monoclonal? the insulin receptor can be cleaved " from the litterature it seems that the insulinreceptor can be cleaved.. Insulin receptor cleavage induced by estrogen impairs insulin signaling BMJ Open Diabetes Res Care. 2021; 9(2): e002467.
Didier Poncet. The expected size of the band is 95kDa. Below three are the nonspecific bands. In the Untreated one, I got my band initially, but when I ran the same as the control, even in untreated I did not get the band, which is quite strange.
Anushka Srivastava. I am dipping a Nitrocellulose membrane in a Transfer buffer containing 20% methanol.
To address the issue of intense non-specific bands in your Western blot analysis for Insulin receptor Beta, consider implementing the following technical strategies:
1. Antibody Titration
Primary Antibody Concentration: Conduct a titration of your primary antibody to identify the optimal concentration that minimizes non-specific binding. High antibody concentrations can lead to increased background noise and non-specific interactions.
2. Blocking Optimization
Blocking Agent: Increase the concentration of BSA beyond 5% or consider alternative blocking agents such as non-fat dry milk, casein, or commercial blocking buffers. This may help reduce non-specific binding by saturating potential binding sites.
3. Washing Protocol Enhancement
Washing Steps: Increase the number of washes during the post-incubation steps. Incorporate Tween-20 (0.05-0.1%) into your wash buffer to enhance the removal of loosely bound antibodies, thereby reducing background signal.
4. Sample Preparation Protocol
Denaturation and Reduction: Ensure that samples are adequately reduced and denatured prior to loading onto the gel. Utilize fresh reducing agents such as DTT or β-mercaptoethanol (BME) and boil samples for 5-10 minutes to ensure complete denaturation.
5. Control Experiments
Negative Controls: Include appropriate negative controls, such as lysates from non-transfected or irrelevant cell lines, to ascertain that observed bands are specific to the Insulin receptor Beta and not due to endogenous proteins or cross-reactivity.
6. Characterization of Protein Isoforms
Isoform Analysis: Investigate potential isoforms or post-translational modifications of the Insulin receptor Beta that may contribute to additional bands. This may necessitate the use of specific antibodies designed to recognize distinct isoforms or adjusting electrophoretic conditions.
7. Antibody Quality Assurance
Antibody Specificity: Confirm that both primary and secondary antibodies are validated for specificity against Insulin receptor Beta. If using polyclonal antibodies, consider switching to monoclonal antibodies, which typically exhibit higher specificity and lower background signal.
Syeda Sadaf Rizvi You can try once with PVDF membrane. But again, to get rid of the non specific binding you have to optimize the blocking step and antibody concentrations.
Thank you, everyone I am going to repeat it by next week, with all your kind suggestions, I hope I will get the strong band this time.
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