
Multiple antibody blotting - western blot
#1
Posted 09 February 2009 - 03:33 PM
i'm planning to incubate the membrane overnight with 3 different antibodies made in the same specie.
the three protein are different in size .. so I don't understand why it shouldn't work
does someone experience with this ?
thank you Gianluca
#2
Posted 10 February 2009 - 02:36 AM
Sometime some antibodies also recognize an other band, it's considered as normal and we don't care (unless if you incubate also with an other antibody that should recognize something close to the non specific band.)
Sometime one of your protein of interest make aggregates, dimers, then you would get two bands with only one antibody, which could also be a problem if you incubate several antibodies. You won't be able to say if the band you see is one of your protein of interest, or one non specific band of an other protein of interest, or some dimers or aggregates of the third protein of interest.
I would do a simultaneous incubation only if I would already have done single incubation and confirmed that I only get one band each time.
#3
Posted 10 February 2009 - 04:38 AM
#4
Posted 13 March 2009 - 09:51 PM
#5
Posted 26 March 2009 - 12:46 PM
#6
Posted 16 April 2009 - 05:50 AM
Edited by yobou, 16 April 2009 - 05:51 AM.
#7
Posted 01 July 2009 - 06:03 AM
i have used max 4 antibodies in a single membrane once, giving me only 4 specific bands.
this works with mouse antibodies from BD biosciences, but did not work with rabbit antibodies from cell signaling. would not recommend this approach with goat antibodies from santa-cruz

Edited by rajgene, 01 July 2009 - 06:09 AM.
#8
Posted 02 July 2009 - 04:27 AM
i dont think why people should be worried of this technique
you answered your own question when you listed a random bunch of antibodies not to do it with - its an invalid technique as it is based on dumb luck - antibody/antibody interactions are seriously unpredicatable and best avoided
d
#9
Posted 02 July 2009 - 07:19 AM
#10
Posted 06 July 2009 - 01:49 PM

P.S in my previous post i just hinted my experience with different antibody sources based on the quality of antibodies provided by the manufacturer.(BD and cell signaling being the best)
Edited by rajgene, 06 July 2009 - 02:02 PM.
#11
Posted 06 July 2009 - 11:16 PM
You have to be careful, and keep the mix antibodies only to repeat a already known result.
#12
Posted 08 July 2009 - 07:53 AM
Also, I routinely do this with secondary antibodies as well. Sometimes the same species, sometimes not (depending on the primary, obviously). Other times to test different isotypes, i.e. IgM + IgG secondary antibodies mixed (I regularly screen for hybridoma positives). Occasionally, I do get increased background when mixing the secondary antibodies so I either wash longer or dilute them back a little more than usual.
Although it is best if you don't have to mix antibodies, sometimes the samples you are running are limited so this is definitely a possible alternative. I also cut membranes if possible, but I don't wrap them I simply take a pair of scissors and cut. Sometimes horizontal to separate bands/antibodies by size and sometimes vertically so separate multiple sets of the same thing run on a single gel.
If you have enough antibody, it's worth trying. If you don't like what you see, you can always go back and do them separately.
#13
Posted 15 September 2009 - 10:48 PM
interactions of antibodies is not seen only in cocktails, but also after an incomplete stripping of one antibody followed by reprobing with another in WB where a potentially misleading bands can be found on the membrane
Hi there?
"stripping?" what's that exactly. Requires special buffer or just blocking buffer overnight will do???
thanks
#14
Posted 24 September 2009 - 05:32 AM
interactions of antibodies is not seen only in cocktails, but also after an incomplete stripping of one antibody followed by reprobing with another in WB where a potentially misleading bands can be found on the membrane
Hi there?
"stripping?" what's that exactly. Requires special buffer or just blocking buffer overnight will do???
thanks
requires special buffer (beta-mercaptoethanol, pH changes...), to cleave the antibodies bound.
#15
Posted 25 September 2009 - 03:30 AM
Well, if you have two antibodies with different secondary you can just do it secuencially.Just remember to add Sodium Azide to your antibody to remove the previous HRP activity
Edited by laurequillo, 25 September 2009 - 03:32 AM.
"This is SPARTA!"
"I´m the goddamn batman"