Why do we transfer proteins from a gel to a membrane to continue with immunoblot
ID: 203755 • Letter: W
Question
Why do we transfer proteins from a gel to a membrane to continue with immunoblotting instead of blotting on the gel itself? What is the difference between nitrocellulose and PVDF membranes and why is methanol ( or ethanol) used in the transfer buffer? Why do we transfer proteins from a gel to a membrane to continue with immunoblotting instead of blotting on the gel itself? What is the difference between nitrocellulose and PVDF membranes and why is methanol ( or ethanol) used in the transfer buffer?Explanation / Answer
In western blotting technique the role of blot is to immobilize protein over them after that antibody specific to protein of interest will bind. In case of gel the protein can’t get immobilized properly and there are chances that protein may be diffused and spread out through pores. That will not make possible to bind at specified site. Second thing is preparation of gel; those would not be prepared in protein free environment. This will be responsible for non specific binding. Blotting helps in fixing this problem and provides the specific and reliable results.
PVDF membrane has higher protein binding capacity while nitrocellulose has less binding capacity in comparison to PVDF membrane.
PVDF have higher sensitivity to detect protein while nitrocellulose has less sensitivity. This property provide the clue that high sensitive will have high noise in background.
In case of PVDF membrane the protein molecule bind through hydrophobic and dipole interaction with antibodies. While in case of nitrocellulose there are only hydrophobic interactions.
Nitrocellulose membrane is suitable for detection of low molecular weight protein and PVDF are suitable for detection of high molecular weight proteins.
The main reason is the hydrophobic nature of buffers, this will not penetrate in membrane and because of this the protein will not bind and transfer significantly.
Soaking in methanol for 15 to 30 seconds will hydrate the membrane and protein will transfer & bind efficiently.
Related Questions
drjack9650@gmail.com
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.