co-immunoprecipitation - (Sep/19/2006 )
how can i isolate the targeted protein from the immuno-complex after co-immunoprecipitation?
-zhouwuchina-
QUOTE (zhouwuchina @ Sep 19 2006, 05:43 PM)
how can i isolate the targeted protein from the immuno-complex after co-immunoprecipitation?
do you want to separate co-immunoprecipitated proteins? or separate protein from the antibody?
I'm not sure to understand.
-behappy736-
after co-immunoprecipitation,we get the product of immuno-complex .how can i seperate this complex without distroying these two proteins?
-zhouwuchina-
QUOTE (zhouwuchina @ Sep 20 2006, 07:22 AM)
after co-immunoprecipitation,we get the product of immuno-complex .how can i seperate this complex without distroying these two proteins?
I didn't understand what you mean. Do you want to separate the complex from the Ab without degrading the proteins? If yes, just use Laemmli buffer, vortex shortly and boil. Do you want to have the whole complex on a gel, i.e. all teh proteins together without separating one from the other? Or you want to separate them from each other and see single proteins?
If you want to see the whole complex, you need to do a native PAGE. If you want to separate them from each other, after the addition of Laemmli you can run a SDS-PAGE.
Please clarify.
-dnafactory-
use low pH, but may denaturate your protein, or high MgCl2 if ionic strength won´t disturb; look for standard protocols to elute proteins from Ab affinity columns...
-The Bearer-
QUOTE (dnafactory @ Sep 20 2006, 04:52 PM)
QUOTE (zhouwuchina @ Sep 20 2006, 07:22 AM)
after co-immunoprecipitation,we get the product of immuno-complex .how can i seperate this complex without distroying these two proteins?
I didn't understand what you mean. Do you want to separate the complex from the Ab without degrading the proteins? If yes, just use Laemmli buffer, vortex shortly and boil. Do you want to have the whole complex on a gel, i.e. all teh proteins together without separating one from the other? Or you want to separate them from each other and see single proteins?
If you want to see the whole complex, you need to do a native PAGE. If you want to separate them from each other, after the addition of Laemmli you can run a SDS-PAGE.
Please clarify.
What i mean is how to seperate the proteins of complex after co-immunoprecipitation. Co-immunoprecipitation is different with immunoprecipitation.The products of the immunoprecipitation are targeted proteins and their antibody,but we get the complex which include the antibody ,targeted protein and the protein binding on them after Co-immunoprecipitation. Maybe these binding proteins are a kind of ligands or receptors and i don't care about them. So my original intention is seperating these binding proteins before 2-D gel.If so,it will be help for the next analysis of MS. clear?
-zhouwuchina-
OK, so you want to do an IP (and not a co-IP), and you want it to be clean.
wash a lot (5 times 5 min) in your RIPA buffer but increase the NaCl up to 0.5M, or even 1M if 0.5M is not enough, during the washing step.
This should help to get rid of protein interactions, but antigen-antibody interaction should stay.
-Missele-
QUOTE (Missele @ Sep 21 2006, 12:39 AM)
OK, so you want to do an IP (and not a co-IP), and you want it to be clean.
wash a lot (5 times 5 min) in your RIPA buffer but increase the NaCl up to 0.5M, or even 1M if 0.5M is not enough, during the washing step.
This should help to get rid of protein interactions, but antigen-antibody interaction should stay.
wash a lot (5 times 5 min) in your RIPA buffer but increase the NaCl up to 0.5M, or even 1M if 0.5M is not enough, during the washing step.
This should help to get rid of protein interactions, but antigen-antibody interaction should stay.
Firstly i am doing co-IP not IP,secondly you mean that the concentration of Na+ can influnce the bond between proteins? it is so effective that it can identify the interaction of proteins and the antibody-antigen even if the two kinds of binding both are the results of hydrophobic bond?
-zhouwuchina-
QUOTE (zhouwuchina @ Sep 20 2006, 05:27 PM)
QUOTE (dnafactory @ Sep 20 2006, 04:52 PM)
QUOTE (zhouwuchina @ Sep 20 2006, 07:22 AM)
after co-immunoprecipitation,we get the product of immuno-complex .how can i seperate this complex without distroying these two proteins?
I didn't understand what you mean. Do you want to separate the complex from the Ab without degrading the proteins? If yes, just use Laemmli buffer, vortex shortly and boil. Do you want to have the whole complex on a gel, i.e. all teh proteins together without separating one from the other? Or you want to separate them from each other and see single proteins?
If you want to see the whole complex, you need to do a native PAGE. If you want to separate them from each other, after the addition of Laemmli you can run a SDS-PAGE.
Please clarify.
What i mean is how to seperate the proteins of complex after co-immunoprecipitation. Co-immunoprecipitation is different with immunoprecipitation.The products of the immunoprecipitation are targeted proteins and their antibody,but we get the complex which include the antibody ,targeted protein and the protein binding on them after Co-immunoprecipitation. Maybe these binding proteins are a kind of ligands or receptors and i don't care about them. So my original intention is seperating these binding proteins before 2-D gel.If so,it will be help for the next analysis of MS. clear?
What you want to do is to elute the protein complexes from the Ab? You want to run your protein but you don't want to have the Ab in the eluate?
-dnafactory-
to dnafactory:
what i want to do as follows:
step 1:
elute the protein complexes from the Ab,
step 2:
seperate the intersting protein from the protein complex.
step 3:
run 2-D gel and analysis of MS
my question is how to complete the first two step without destroying the structure and function of the intersting protein before the Step 3.
-zhouwuchina-