Immunoblot Analysis

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

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Gallagher, S., Chakavarti, D. Immunoblot Analysis. J. Vis. Exp. (16), e759, doi:10.3791/759 (2008).

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

  1. 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...

    Reply
    Posted by: Anonymous
    June 30, 2008 - 3:12 AM
  2. 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.

    Reply
    Posted by: Anonymous
    August 1, 2008 - 6:00 AM
  3. Is there a video for co-immunoprecipitation???

    Reply
    Posted by: Anonymous
    September 14, 2008 - 4:52 AM
  4. How do you quantify the blots with Image-J

    Reply
    Posted by: Anonymous
    October 18, 2008 - 3:48 PM
  5. 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?

    Reply
    Posted by: Anonymous
    December 7, 2008 - 9:43 AM
  6. 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?

    Reply
    Posted by: Anonymous
    January 26, 2009 - 2:48 PM
  7. 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

    Reply
    Posted by: wilton R.
    January 26, 2009 - 3:02 PM
  8. 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...

    Reply
    Posted by: Anonymous
    February 19, 2009 - 11:39 AM
  9. Dr. Gallagher or Dr. Chakavarti! Could I contact you by E-mail directly, please?

    Reply
    Posted by: Anonymous
    April 16, 2009 - 2:10 AM
  10. Please contact me at seang@uvp.com   Best regards, Sean Gallagher

    Reply
    Posted by: Anonymous
    May 1, 2009 - 8:14 PM
  11. in which step can we leave the system overnight else?

    Reply
    Posted by: Nik N.
    April 16, 2009 - 4:45 AM
  12. 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?

    Reply
    Posted by: Nik N.
    April 16, 2009 - 5:54 AM
  13. or optimization of a multiplex (two primary antibodies together) quantitative western using the LI COR Odyssey system?

    Reply
    Posted by: Anonymous
    June 3, 2009 - 10:23 AM
  14. How, a wonderful. I need this article to help my friends (include me, he...he..) in research
    http://infosains.com

    Reply
    Posted by: Anonymous
    July 27, 2009 - 10:39 AM
  15. how I get this video

    Reply
    Posted by: Anonymous
    August 31, 2010 - 4:47 AM
  16. Please email us at support@jove.com

    Reply
    Posted by: Anonymous
    August 31, 2010 - 10:12 AM

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