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Immunoprecipitation


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12 replies to this topic

#1 Danny Chow

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Posted 06 August 2009 - 01:13 AM

Hi, do you guys have some good protocol for IP ?

What i was trying to do is using an antibody to trap the target protein. The following is what i did.

1) Incubate the antibody with the cell lysate
2) remove the unbind cell lysate
3) Add Protein A and incubate
4) Elution

The question that i have , is that i am having a hard time in separating my antibody and my target protein
in the elution fraction, it contains my antibody and also my target protein.

help ....

thanks

#2 little mouse

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Posted 06 August 2009 - 01:47 AM

sorry, it's a really brief description of your protocol and I don't get it. You incubate your cell lysate with antibody and then you remove your unbound cell lysate? How can you do that? is your antibody already bond on beads? I don't get your protocol. Give more details if you want some help.

What I do is:
incubate the sample with antibody
incubate the sample + antibody + protein A coupled to beads
wash
elute by boiling with loading buffer

I get my protein and the antibody used for the IP into the gel.
If I don't want to see the antibody while performing the western blot, I don't use an HRP-anti-IgG, but I rather use HRP-protein A to detect the primary antibody. This will not bind to denatured immunoglobulin used for IP

#3 Curtis

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Posted 06 August 2009 - 02:03 AM

IP is tricky, but it's not hard....

you just need two primary antibodies raised in different species if your problem is how to detect the protein in the western blot part.

for example, IP with ab raised in mouse
and then wb with same ab but raised in rabbit, followed by using secondary ab against rabbit.

Curtis

#4 little mouse

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Posted 06 August 2009 - 03:06 AM

IP is tricky, but it's not hard....

you just need two primary antibodies raised in different species if your problem is how to detect the protein in the western blot part.

for example, IP with ab raised in mouse
and then wb with same ab but raised in rabbit, followed by using secondary ab against rabbit.

Curtis


not working with all antibodies. I had already some cross-reaction between mice and rabbit antibodies.
I was very happy with HRP- protein A

#5 Danny Chow

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Posted 06 August 2009 - 07:06 PM

sorry, it's a really brief description of your protocol and I don't get it. You incubate your cell lysate with antibody and then you remove your unbound cell lysate? How can you do that? is your antibody already bond on beads? I don't get your protocol. Give more details if you want some help.

What I do is:
incubate the sample with antibody
incubate the sample + antibody + protein A coupled to beads
wash
elute by boiling with loading buffer

I get my protein and the antibody used for the IP into the gel.
If I don't want to see the antibody while performing the western blot, I don't use an HRP-anti-IgG, but I rather use HRP-protein A to detect the primary antibody. This will not bind to denatured immunoglobulin used for IP


Will the boiling denatured my target protein ?

#6 Danny Chow

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Posted 06 August 2009 - 07:41 PM

i found so many protocol online, they all said that after the elution, boil the sample to the SDS page or Western Blot to analysis the result.

But is there a way to separate the target protein and the antibody ?
Because i have to use the pure product to test for antiviral effect...

Thanks

#7 little mouse

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Posted 06 August 2009 - 10:38 PM

i found so many protocol online, they all said that after the elution, boil the sample to the SDS page or Western Blot to analysis the result.

But is there a way to separate the target protein and the antibody ?
Because i have to use the pure product to test for antiviral effect...

Thanks


I see, you are purifying the protein.
you have to use a column with covalently bond antibodies. Don't look for IP protocols, it's not the same purpose (in these protocols they use few amount and load all what is IPed on a SDS-PAGE. You would have to look for immunoaffinity purification.
I don't have my lab book here right now. ... I'll try to find a protocol.

#8 little mouse

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Posted 06 August 2009 - 11:03 PM

Depending on your antigen-antibody interaction, you might try different elution conditions.
Have a look here

http://www.piercenet...BA-8DFBC1B802D3

#9 little mouse

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Posted 06 August 2009 - 11:09 PM

And here you would find some information about the agarose :

http://www.piercenet...?fldID=01020201

(I was using CnBr agarose.)

#10 Danny Chow

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Posted 07 August 2009 - 12:04 AM

And here you would find some information about the agarose :

http://www.piercenet...?fldID=01020201

(I was using CnBr agarose.)


I tried this before, but it failed to capture anything... plus... i am expressing this protein in mammalian cell ... the yield is very low.. the only method for detecting this protein is western blot... so troublesome ..
But anyways, thank you for the advice !
i think i will try it one more time by using this immunoaffinity chromotography

#11 little mouse

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Posted 07 August 2009 - 12:55 AM

What antibody did you use for capture? one for ELISA or flow cytometry, immunohistochemistry? you must take something directed against the native form of the protein, and not against a denatured form as it is often the case in Western-blot.

#12 Danny Chow

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Posted 09 August 2009 - 09:34 PM

What antibody did you use for capture? one for ELISA or flow cytometry, immunohistochemistry? you must take something directed against the native form of the protein, and not against a denatured form as it is often the case in Western-blot.


let me tell you the whole story....
what i did is using my antibody, and mixing with the sample, after that, i added Protein A inside the mixture of my sample and antibody...
after doing the elution, i just run my elution fraction in SDS PAGE and Western blot with the antibody in it, therefore, i put my antibody in one of the lane in SDS PAGE and also Western blot as a control. The result showed that there is something that after IP, appeared in the elution fraction but not the antibody lane.
Then my Boss told me to separate the antibody and the additional band showed in Western blot...

I think that the antibody is ok, since it can capture the right thing for me, but just i don't know a way to separate the antibody and my target protein after IP ...

#13 Kristina @Invitrogen Dynal

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Posted 09 September 2009 - 05:27 AM

To prevent antibody from being co-eluted with your target protein, this antibody must be covalently coupled to the solid support you are using. As you only have an small amount of target protein in your sample, you will need an efficient solid support to make the capture work. Dynabeads are small and numerous - giving them a much higher 'availability', i.e. binidng kinetics compared to sepharose/agarose products.

You can use Dynabeads Protein A or G for the capture, see www.invitrogen.com/immunprecipitation and crosslink the antibodies to the beads. See method here: www.invitrogen.com/crosslinking

Another alternative is to couple your primary antibody directly to Dynabeads M-280 Tosylactivated. The antibody will be covalently coupled to the surface, so it will not co-elute.

I hope this helps

Kristina




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