Multiple antibody blotting - western blot - (Feb/09/2009 )

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.

yobou on Jul 3 2009, 12:19 AM said:

Hi there?
"stripping?" what's that exactly. Requires special buffer or just blocking buffer overnight will do???

thanks

mrcellline on Sep 16 2009, 08:48 AM said:

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

gianluca on Feb 9 2009, 04:33 PM said:

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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