Biology
Summary
Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.
Cite this Article
Copy Citation | Download CitationsGallagher, S., Chakavarti, D. Immunoblot Analysis. J. Vis. Exp. (16), e759, doi:10.3791/759 (2008).
Translate text to:
Choose Language…
Abstract
Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.Protocol
The complete text protocol for this experimental approach is available in Current Protocols in Molecular Biology
Disclosures
The authors have nothing to disclose.
Comments
16 Comments
Post a Question / Comment / Request
You must be signed in to post a comment. Please sign in or create an account.
hello, for extraction of proteins from cell if we want to lyse whole cell we should use antiprotease .Could we use only PMSF for this purpose or we must use also other antiprotease with it...if our desired protein is chemokine that secretes to supernatant of our cell culture flask how could we extract it ....thanks alot for your kinds...
It would be great if people submit discussing the troubleshooting of western blots (one of such kind has been dealt in this video..air bubbles trapped in western blots..coz the blotting membrane was put very rapidly in to the transfer buffer for pre wetting) Post them! Sudheendra.
Is there a video for co-immunoprecipitation???
How do you quantify the blots with Image-J
i try to do dot-blot but after using lysis buffer when i lay 1 microlitter of it on PVDF membrane pvdf was lysed what should i do?
I didn't see you wash after the milk blocking. You used the antibody right after. Is this how you really do it, or did I miss a step?
Couple questions Did you do a wash after the milk blocking? or did I miss something? I am tring to download the pdf..for somereason it is saving me in a very strange format that my pc dŒsn't recognize. Can u explain? THanks WIL
You do not have to wash after blocking. You can block then put in primary overnight. After primary and after secondary is where you have to wash so you dont have a lot of background. Works all the time for me...
Dr. Gallagher or Dr. Chakavarti! Could I contact you by E-mail directly, please?
Please contact me at seang@uvp.com Best regards, Sean Gallagher
in which step can we leave the system overnight else?
At the end of procedure when we dye the Immobilon-P membrane with BioRad 1immu-StarTM AP Chemo luminescent Protein Detection System: Do we have to remove excess of dye from membrane just before luminescent detection?
or optimization of a multiplex (two primary antibodies together) quantitative western using the LI COR Odyssey system?
How, a wonderful. I need this article to help my friends (include me, he...he..) in research
http://infosains.com
how I get this video
Please email us at support@jove.com