Methods and Techniques Discussion => Western Blotting (WB) => Topic started by: jbiedenkapp on May 13, 2005, 11:47:21 AM

Title: re-cycling/re-using primary antibodies for Western Blot
Post by: jbiedenkapp on May 13, 2005, 11:47:21 AM
was wondering if there were any standards as to how many times primary antibodies can be re-used for Western blot.  does this depend on the antibody used, concentration, etc...  we are interested in actin, c-fos, zif-268, arc.  if anyone has experience with any of these primaries it would be helpful to know how many, if any, times they can be re-used and still get reliable staining.
thank you
Title: re-cycling/re-using primary antibodies for Western Blot
Post by: ImmunoNYC on June 02, 2005, 12:31:44 AM
Although highly undesirable for the sake of reproducibility I have reused antibodies quite a bit. I don't have experience with those specific Abs you mention but I know I have often reused antibodies for westerns a bunch of times, especially when they are ultra precious antibodies. I think the concern is often to make sure nothing nasty grows in them during storage more than anything else.

Also, interestingly, I have in truly desperate situations reused antibodies even for IHC (when doing dunking batch method of staining in coplin jars). Again not ideal, but it does work.
Title: Re: re-cycling/re-using primary antibodies for Western Blot
Post by: richard03 on September 27, 2007, 10:55:08 PM
I used to re-use antibodies for IHC staining. In average, I re-use diluted antibodies for about 10 times. The 4th-6th times seem perform better than other times.

 
Title: Re: re-cycling/re-using primary antibodies for Western Blot
Post by: vivfly on September 29, 2007, 06:34:08 AM
I used to re-use antibodies for IHC staining. In average, I re-use diluted antibodies for about 10 times. The 4th-6th times seem perform better than other times.

 

I was told that re-used antibodies will give less background, but I haven't figured out how to re-collect the antibodies? For some antibodies, it is suggested to incubate for O.N at 4 oC. What i could do is to cover the sections with coverslips in order to avoid of drying, but seems impossible to re-cycle the antibodies the next day. 
Title: Re: re-cycling/re-using primary antibodies for Western Blot
Post by: richard03 on September 29, 2007, 02:56:17 PM
I re-collected antibodies when I did brain sections free floating incubation.

If your sections are on slides, you may not coverslip the sections, but the slides should be put in humidified chambers and incubate at 4 C to avoid drying. Then you can re-collect the antibody using a pipette.
Title: Re: re-cycling/re-using primary antibodies for Western Blot
Post by: vivfly on September 30, 2007, 02:49:32 PM
I re-collected antibodies when I did brain sections free floating incubation.

If your sections are on slides, you may not coverslip the sections, but the slides should be put in humidified chambers and incubate at 4 C to avoid drying. Then you can re-collect the antibody using a pipette.


Thanks a lot :)