Hello,

If the loading buffer for Western blot is expired, migration and pattern/shape of protein bands might be affected, because the buffer contains SDS (a denaturing agent) that might degrade after the buffer expires, resulting in incomplete denaturation of the proteins and migration efficiency of proteins on gel.

Furthermore, the pH of the buffer may also shift that in turns, affect the stability and charge of the proteins. As a consequence, this may alter protein migration patterns and their stability.

Also, loading buffer often contains β-mercaptoethanol to maintain protein stability and to prevent protein aggregation. Over the time, the agent might become unstable, cause degradation or aggregation of proteins.

I hope this information is useful for you.

Good luck!

Phan Thi Lam Hong thank you so much doctor for your response. I am struggling from last 4 months but i didn’t notes that . And now i 100% sure that loading buffer was the problem and changed and confirmed it twice and get the results. Again appreciated your response

I'm truly thrilled to hear about your results! Keep up the excellent work, and may you achieve even greater success in the future!