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Heavy and light Chain

IP and CO IP

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9 replies to this topic

#1 ulujm

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Posted 11 May 2014 - 12:50 PM

Hi

 

I am starting an IP experiment.

Here it is.

 

I want to check interaction betweein human protein X and Human protein Y.

 

I did IP the ProtX with mouse AntiX, and proein A bead.Then I did my western  with the mouse antiX as a control and mouse anti Y (first antibody) to check if I have interaction..

Ifinally I use an antimouse HRP (2nd antibody) for ECL detection.

 

I have a few question.

 

Concerning the Heavy and light chain,  what antibody are they coming from? are they coming from the antiboy I use to ip (mouse antiX) or the first antiboy( mouse anti Y).

 

what is the bet way to get read of the HC and Lc. I heard to pick difference species of antibodies what is that?

 

Anothe matter: if Y and X interact , will the antiX able to puldown X since Xinteract with Y so the epitope will not be available.

 

thanks



#2 bob1

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Posted 11 May 2014 - 12:56 PM

The HC and LC bands you see are coming from the antibody used to precipitate your protein - if they were from the antibody you used to detect the proteins you wouldn't get a band on the gel you would just get a lot of background.

 

To get around this problem you need to use a detecting antibody from a different species - e.g. if you IP with a mouse antibody, use a rabbit antibody to detect the protein.

 

It may be the case that X and Y interaction can't be seen if Y is masking the epitope.  You should make sure that your antibody is validated for IP.  You can test this by getting some tagged protein (e.g. FLAG, HA, V5, GFP etc.) and IP with your antibody, then try detecting with an antibody against your tag.



#3 ulujm

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Posted 11 May 2014 - 01:28 PM

Hi hanks

 

So

 

If I IP with mouse antiX, I western with my mouse AntiY, then I should detect with an rabbit anti mouse HRP. or a mouse anti rabbit HRP  r?

thanks



#4 bob1

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Posted 11 May 2014 - 08:09 PM

No, IP with mouse anti-X, detect with rabbit anti-Y (and/or rabbit anti-X) then use appropriate secondary (usually goat anti-rabbit HRP or similar).



#5 ulujm

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Posted 12 May 2014 - 02:27 AM

ok sorry I thought thet you were talking about the HRP antibody when you weretalking about the detection antiboy, you mean the first antibody,

 

 

so,  IP with mouse antiX, then  first antibody with Rabbit antiY, 2nd antibody goat antirabbit HRP. Also since I want to check if my X ip was working I should use as well an rabbit antiX.

 

correct?



#6 bob1

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Posted 12 May 2014 - 01:08 PM

Yes - that's the ideal situation and should prevent you seeing the IgG bands on your membrane.  Another solution is to use allows you to use the same type of antibody as you used for the IP (e.g. IP with mouse anti-X and detect with mouse anti-X or Y) is to have a protein G-HRP conjugate in the place of your secondary antibody.  This will not detect the denatured IgG which has been run with your IP samples.



#7 ulujm

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Posted 12 May 2014 - 01:29 PM

ok thanks

 

 

ProteinA-hrp will work as well.. Maybe I should do. cheaper than buying more antibodies



#8 ulujm

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Posted 15 May 2014 - 06:41 AM

hey

 

Can I do a transfer after a comassie staining.?



#9 bob1

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Posted 15 May 2014 - 01:41 PM

Depends on the coomassie stain - most fix the gel so the proteins won't transfer properly.  It is usually best to run two gels, transfer one, stain the other.



#10 mdfenko

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Posted 16 May 2014 - 03:43 AM

hey

 

Can I do a transfer after a comassie staining.?

in general, yes. the fixation with coomassie stains (usually with acetic acid) is reversible. addition of 0.05% sds to the transfer buffer is recommended.

 

but, some of the coomassie will be released from the protein and may give a false sense of transfer. extended transfer times will be required to ensure sufficient transfer.


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