Phosphoantibodie get better and better after every incubation - (Mar/18/2011 )

Hi everybody,

I need some help. I try to detect my phospho antibodies. After the first incubation an do not see them, but if I do another antibodie on the membran then there will appear an band at the size of my first phospho antibodie. The bands are no extra bands from the second antibodie that is sure. So can somebody please explain that to me or did some one make the same observations?

Could you describe your assay more precisely? Is that a WesternBlot? What is the protein you are assessing? Do you use only 1 antibody?

magic western on Fri Mar 18 08:50:55 2011 said:

Is your secondary Ab used for phospho Ab working ?
I would suggest to just add in 1 epi. 1 µl of it along with westernblot substrate, and check for chemiluminescence in dark with eyes.
Cheers,

yes I do western blot. 12%SDS Gel, semi dry blot, first antibody (phospho) over night at 4 Celsius, secondary antibody HRP linked for 1h, ECL reaktion 1 min, detection. My secondary antibody is rabbit HRP antibody. Datasheets suggest them. Then I can´t detect my phospho antibody for example p-p70. But on the next day lets say I put p-p42/44 on the membran over night, then second antibody, ECL reaktion, detection. I will see my p-p42/44 and also a band up to 70. Is this my p-p70 from the day before?

@ Date Rahul: I don´t get your idea. You suggest that I should take some µl of my protein and add 1µl Antibody (first or second?) to it and check for luminescence? But why?

magic western on Fri Mar 18 12:50:49 2011 said:

I suggested that just to check your Secondary is still functional(HRP). Just by adding it with substrate.
what is your secondary for pp42-44 ?

Do you see only one additional band around 70 kd or more after pp42-44 incubation?

I have a few questions for you: Are you stripping your blot before incubation with your second phospho antibody? How are you washing and blocking? Is your secondary antibody the same for both primaries?

I think what's happening is your second antibody is reacting with your anti-p-p70 as well as your p-p42/44. As for why you see stronger signal second time round, maybe 1h wasn't enough in the first incubation. Also, if you are not stripping the membrane, on your second day p-p70 will have more secondary bound to it than on the first day, hence the brighter signal.

magic western on Fri Mar 18 12:50:49 2011 said:

What Date Rahul suggests is that you check that the HRP on your secondary antibody is still functional, as well as your ECL. All you have to do is put 1ul of your secondary antibody in a tube, add ECL reagent, and look for light. Do this in the dark room, if all reagents are fine you should be able to see light with the naked eye.
From your post I don't think this is your problem thou, as you said you see signal on the second day, but is definitely an easy way to check your reagents

Hi almost a doctor,

that really sounds like a possible answer. No I do not stripp my membran, I wash with TBS/T and block in milk TBS/T and yes my secondary is for both primaries. So you also thing that the band at around 70 is the p-p70 band form the day before? Because that is what I thing too. So I should be able to check this very easyly if I will incubate the second ab for p-p70 a bit longer and maybe with more antibody? That sounds really great. Thanks a lot.