Western blot for two proteins: any alternative to stripping? - (May/08/2006 )
Hi there,
I want to do a ECL-Western for desired Protein (23kD) and for Actin (42kd) as loading control.
Normally I would just incubate with both primary antibodies at the same time.
Unfortunately my 23kD-antibody is very unselective so I get signals with it at 42kD, too.
I would like to avoid stripping and reprobing because of protein loss (a third -
chromogenic incubation is run afterwards).
Is there any possibility to "quench" the signal of the first inkubation besides stripping?
Thanx
Kylvalda
P.S. Is it possible to perform a ECL-Detection after a chromogeneic Detection with the same blot?!?
I want to do a ECL-Western for desired Protein (23kD) and for Actin (42kd) as loading control.
Normally I would just incubate with both primary antibodies at the same time.
Unfortunately my 23kD-antibody is very unselective so I get signals with it at 42kD, too.
I would like to avoid stripping and reprobing because of protein loss (a third -
chromogenic incubation is run afterwards).
Is there any possibility to "quench" the signal of the first inkubation besides stripping?
Thanx
Kylvalda
P.S. Is it possible to perform a ECL-Detection after a chromogeneic Detection with the same blot?!?
if your 23 kDa antibody is very unselective, why wouldn't you first detect the 42 kDa, and then detect the 23 kDa, without stripping?
or you could simply cut the blot between 23 and 42 kDa and incubate with the appropriate antibody each half-blot.
I never tried ECL after chromogenic, but I don't see a good reason for not working. (keep in mind that you will detect the antibodies used for the chromogenic reaction during ECL detection)
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(keep in mind that you will detect the antibodies used for the chromogenic reaction during ECL detection)
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Are you sure?
The chromogenic reaction is Alkaline Phosphatase-linked and shouldn't convert luminol as substrate in the ECL-reaction...
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if your 23 kDa antibody is very unselective, why wouldn't you first detect the 42 kDa, and then detect the 23 kDa, without stripping?
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Very good idea - I'll try that!
If I want to do a chromogenic detection AFTER an ecl-detection, it seems to me stripping isn't necessary because horseradish-peroxidase shouldn't react with nbt/bcip, right?
keep in mind that there are already primary antibodies bound to the membrane and that the secondary antibodies might bind to them.
keep also in mind that some cross reactivity can also occur.
example :
- mouse anti protein X
-anti-mouse-HRP
and then you want to see an Y protein by chromogenic method
-you bind mouse anti protein Y on a blot where there is already mouse antiprotein X
-you add anti-mouse-AP which will recognize anti protein Y but also antiprotein X.
keep also in mind that some cross reactivity can also occur.
example :
- mouse anti protein X
-anti-mouse-HRP
and then you want to see an Y protein by chromogenic method
-you bind mouse anti protein Y on a blot where there is already mouse antiprotein X
-you add anti-mouse-AP which will recognize anti protein Y but also antiprotein X.
So this is my plan:
First detection: 1.AK:anti-actin from rabbit; 2.AK: anti-rabbit ECL
Second detection: 1.AK: anti-cytoglobin from rabbit, 2.AK: anti-rabbit ECL
Third detection: 1.AK: anti-myoglobin from goat, 2.AK: anti-goat chromogenic
Sounds like a good plan to me...
keep in mind that there are already primary antibodies bound to the membrane and that the secondary antibodies might bind to them.
keep also in mind that some cross reactivity can also occur.
example :
- mouse anti protein X
-anti-mouse-HRP
and then you want to see an Y protein by chromogenic method
-you bind mouse anti protein Y on a blot where there is already mouse antiprotein X
-you add anti-mouse-AP which will recognize anti protein Y but also antiprotein X.
So this is my plan:
First detection: 1.AK:anti-actin from rabbit; 2.AK: anti-rabbit ECL
Second detection: 1.AK: anti-cytoglobin from rabbit, 2.AK: anti-rabbit ECL
Third detection: 1.AK: anti-myoglobin from goat, 2.AK: anti-goat chromogenic
Sounds like a good plan to me...
I bet a smile that your anti-rabbit is a goat antibody.
then, your anti-goat will recognize it.
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I bet a smile that your anti-rabbit is a goat antibody.
then, your anti-goat will recognize it.
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No, it's from donkey 
well, why don't you do 2 membranes?
Don't have enough protein - and I thought if I always use the same membrane I can exclude pipetting errors...