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Indirect vs Direct immunoprecipitation


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#1 Hege

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Posted 15 February 2010 - 01:51 PM

Hi.
Ive been doing IPs for a while using monoclonal antibodies and Invitrogen magnetic beads. I have been using the direct method with incubating the beads with the ab overnight, followed by an overnight incubation with the cell lysate. I now want to try using the indirect method, but i do not know how long to incubate the ab+lysate, and then the Ab-Lysate-beads. Has anyone got any experience with this? Help much needed and greatly appreciated! :) Im trying to pull down a membrane protein that i have overexpressed-am then interested in discovering interacting proteins. :P

#2 laurequillo

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Posted 16 February 2010 - 08:00 AM

Hi.
Ive been doing IPs for a while using monoclonal antibodies and Invitrogen magnetic beads. I have been using the direct method with incubating the beads with the ab overnight, followed by an overnight incubation with the cell lysate. I now want to try using the indirect method, but i do not know how long to incubate the ab+lysate, and then the Ab-Lysate-beads. Has anyone got any experience with this? Help much needed and greatly appreciated! :) Im trying to pull down a membrane protein that i have overexpressed-am then interested in discovering interacting proteins. :(


Using proteinA/agarose Ive done the Ips in different ways.
1-Ive left the Ab for 2h and then the beads for 1h;
2-The Ab o/n and the beads 2h;
3-and the Ab 4h and the beads overnight.

All three of them work in my hands, you just have to see if you have background or not (if your Ab works well and you have enough material the first option is enough; with the other two protocols you will increase your background and inespecific binding)

I dont use the magnetic beads, so you have to adjust the time to your beads (I think that beads are quite fast! :) some people in my lab used them and they did the reaction in 1h with the Ab plus 20min with the beads).
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#3 Hege

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Posted 16 February 2010 - 09:20 AM

Hi.
Ive been doing IPs for a while using monoclonal antibodies and Invitrogen magnetic beads. I have been using the direct method with incubating the beads with the ab overnight, followed by an overnight incubation with the cell lysate. I now want to try using the indirect method, but i do not know how long to incubate the ab+lysate, and then the Ab-Lysate-beads. Has anyone got any experience with this? Help much needed and greatly appreciated! :) Im trying to pull down a membrane protein that i have overexpressed-am then interested in discovering interacting proteins. :(


Using proteinA/agarose Ive done the Ips in different ways.
1-Ive left the Ab for 2h and then the beads for 1h;
2-The Ab o/n and the beads 2h;
3-and the Ab 4h and the beads overnight.

All three of them work in my hands, you just have to see if you have background or not (if your Ab works well and you have enough material the first option is enough; with the other two protocols you will increase your background and inespecific binding)

I dont use the magnetic beads, so you have to adjust the time to your beads (I think that beads are quite fast! :) some people in my lab used them and they did the reaction in 1h with the Ab plus 20min with the beads).



Thank you so much. Is there a way to crosslink the ab to the beads after the ab has been incubated with the lysate? I use BS3 to crosslink. Would you reccomend still doing this step?

Hege

#4 laurequillo

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Posted 16 February 2010 - 10:14 AM

[/quote]


Thank you so much. Is there a way to crosslink the ab to the beads after the ab has been incubated with the lysate? I use BS3 to crosslink. Would you reccomend still doing this step?

Hege
[/quote]


I dont crosslink the Ab to the beads, but I guess you should do it before you incubate both with the lysate.

Actually I was reading again what we did in the lab and It was first we incubated the Ab with the beads 20min, then wash the beads 3x, and then you incubate with the lysate for 1h. (Sorry I wrote it in the other way around, but I would say it is not a problem anyway)
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#5 mdfenko

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Posted 17 February 2010 - 07:33 AM

Thank you so much. Is there a way to crosslink the ab to the beads after the ab has been incubated with the lysate? I use BS3 to crosslink. Would you recommend still doing this step?

Hege

thermo-pierce offers a kit for crosslinking antibody to protein a/g for ip. they use disuccinmidyl suberate to crosslink.

crosslink ip kit
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