Hello,

Have you checked from the literature if the surface receptor protein forms oligomer? Add higher conc. of reducing agent like BME or DTT and incubate for sometimes before heat denaturation to break intermolecular disulphide bonds. Are you sure that the negative control was loaded in the first lane? What is the detection method of your western blot samples? Magnetic bead pull down with antibody can be tricky as other interaction partners can be co-immunoprecipitated. The sample should be reduced and denatured well.

Best wishes.

Satyendra Mondal, thank you for your reply.

We have identified a novel surface receptor protein in our lab and used Western blotting to detect it. Our protocol involves using primary and secondary antibodies, with BCIP tablets as the substrate. The first lane of our blot contains a control sample infiltrated only with MME buffer.

Despite performing the Western blot multiple times and testing different membrane-blocking methods (5% skimmed milk and 3% BSA) and varying concentrations of both primary and secondary antibodies, I consistently observed the same band pattern.

Hello,

If the control sample lane has bands, then it is non-specific binding of the antibody. But you have obtained these bands in all lanes (control and test), so these are specific to your sample preparation. You need some modifications in sample preparation (including co-immunoprecipitation). To improve the western blot result, you can follow these tips:

• Dilute the antibodies concentrations,
• Maximize the washing steps and intervals,
• Block the membrane overnight at 4 °C.

okay.

Thank you for your valuable suggestions.