hello to everybody.
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

I would be careful with that.
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.

This could get ugly, fast. If all the sizes are different, why not cut your membrane? I run marker on both sides of the gel, ponceau after transfer and cut the membrane so I can incubate all three of the correct sizes with the desired antibody at the same time but not by mixing the antibodies. Just be sure to block after cutting the membrane so antibody doesn't bind the edge where you cut. I know western blotting is time consuming and everyone wants the answer to their experiment now (ok, only if it's the right answer) but this technique is one that will generally pay off the best with patience. Don't push too fast or with too much antibody (especially mixing them!!) or you'll wind up with a black piece of film.

sequential use of antibodies should be done. Results obtained with antibody cocktails should be invalid.

Hi there, Question to Rkay, you say you transfer, do stain on the blot, then block and then cut. How do you cut the blot (vertically, horizontally?). What do you use to cut the blot?

cutting the membrane can be done vertically and/or horizontally depending upon your loading of samples on the gel. first wrap the PVDF membrane in saran wrap then draw a line with a marker pen on the site of cutting then cut by a sissor. I always do this to detect multiple proteins on the same blot with good signal without the need for antibody coctail, or stripping. In addition it saves time and consumes less amout of antibody (save mony as well)

I do it all the time, provided the antibodies(monoclonal) are really really good. i dont think why people should be worried of this technique its really helpful when you are running out of precious patient samples.
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

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

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

yes of course it has to be standardized for each needs. but i am 100% sure it always works provided you know what your doing. its no dumb luck..all you have to do is just give it a try and save valuable time and money.

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)

It's not only a problem of quality of antibody, you can also have some unexpected results. for example : I was working on two proteins, one of 67 kDa and one of 30 kDa. Both antibodies were perfect, only recognizing one band.But in some conditions, the 30 kDa protein was showing some dimers (bad reducing conditions? I was even using very high concentration of DTT, so I don't know). Then, the question is : is the 60 kDa band the 67 kDa protein or the dimer of 30 kDa ?
You have to be careful, and keep the mix antibodies only to repeat a already known result.

I have mixed primary antibodies (both monoclonal and polyclonal) without much problem for years, but I wouldn't suggest doing it with an antibody you haven't used before since you don't know what to expect.

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.

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.

Normally I cut the membrane and use different antibodies (if possible).If not stripping is ok, but sometimes you destroy the signal.
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

You can, but you must perform the apropiate parallel controls.

One of you samples should by loaded 4 extratimes that will be cutted. One for each antibody and the fourth for the negative control.

Mandatory: as a negative control use unspecific IgG of the same origin as your primary antibody at the same concentration as your primary antibodies. This IgGs are not expensive and very valuable.
If you use a cocktail of primaries sume up the concentrations to obtain the IgG that should go to the negative control. (If the primaries are of differen origin then you must mix IgG from all those origins).
You may-will find yourself shocked with the bands that you supposed good but are unspecific.
This unspecificity come from the primary itself, from the simple fact that it is an antibody no matter which epitope or purification.
The reason is antibodies (IgGs) have disulfur bonds and charged aminoacids.
To avo¡d this extra-bands you can tryto preincubate your antibody with iodoacetic acid or reduced glutatione. this will quench free sulphiydryls. To avoid charges interaction you can ad tween or more salt.

Of course sometimes the antibody contain non-antibody binding stuff (other antibodies or proteins). With primaries there isn't much to do about it but with secondary I'd advice to buy only affinity purified and Fab fragment if possible.

I can incubate all three of the correct sizes with the desired antibody at the same time but not by mixing the antibodies.

Just be sure to block after cutting the membrane so antibody doesn't bind the edge where you cut.


I know western blotting is time consuming and everyone wants the answer to their experiment now (ok, only if it's the right answer) but this technique is one that will generally pay off the best with patience.


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