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Can I incubate membrane with 2 antibodies at the same time ? - (May/07/2008 )

I have #1ab rabbit anti OGG1 ~ 38 kDa and #1ab rabbit polyclinal anti MTH1 ~ 18 kDa ; both I use 1:1000

if I can do that, for the secondary antibody (donkey IgG HRP anti rabbit), should I increase conc. to 1:1000 ? -- normally I use 1:2000


Hi Naka,

Do you run your gel so that these 2 proteins are seperated far apart?

For me, i would always stain my membranes (after transfer) with Ponceau S just to make sure that my proteins seperate nicely. I can also cut my membrane based on the markers so as to block with different antibodies at the same time.

Not sure if this can help you. blush.gif


in theory this is possible, although if you have strange results from an aspecific signal, you don't know which primary Ab caused it, but this happens only in rare cases, so if I were you, I would do the combination

you can keep the 1:2000 dilution of the secondary ab as you do when you only have one antibody, you will still see a clear signal

good luck!


Thank you Chrisgqh and Dpo,

> Do you run your gel so that these 2 proteins are seperated far apart?

No, they aren't, it about 1.5 cm far each other. I'm not sure myself that I will cut correctly, so that I'd like to try both antibodies at same time.

So that mean, if I have #1 antibody from same source (rabbit), I can incubate both at the same time.
On the other hand, if I have #1 antibody from difference source such as rabbit or mouse. I have to incubate membranes separately, do I correct ?


when I have antibodies from two different species, I do the incubations with the primary antibody at the same time, but for the secondary antibody I do it in two steps. Most of the time I first use an HRP coupled secondary antibody for my first species and after visualization of this, I incubate my blots with the second secondary antibody coupled to AP to the second species. This has worked for me in a couple of different settings, but of course you should always be careful when you mix antibodies and only do this with antibodies that you have used seperately before, so you know how specific they are.
Of course, the safest way is to do them separately, but if you want to save some time, combining the primary antibodies can work.


Personally I would do them sequentially and not in parallel. If they are sufficiently seperated you don't have to strip first but I would do...
Primary #1
Seconday #1

Block (?)
Primary #2
Secondary #2


I've had good luck with this, sometimes I even develop after the first one to check if it worked, was briefly, reblock, reprobe and I get both bands