Immunoprecipitation

[Tiffy]

Hello everybody,
is it right that under optimal parameters I got only one band after IP and Western Blot? Today I did my first IP and I got 2 bands but not my band of desire
The bands were at ~ 45-50 kDa and ~ 20 kDa. Is this the destroyed fishing-ab (heavy and light chain)?
It would be very kind if someone could answer my question.
Thanks
Tiffy

[moleculardude]

Hey Tiffy!, unfortunately it seems that it may be your antibodies. Which species did you use? and which types (specificity)?

reduced and denatured rabbit IgG heavy chain band runs at ~ 50 kDa
close to your size....

also 25 kDa may be the other antibody fragment...

please post the species info and your protein of interest and we may help more

cheers!

Originally Posted by Tiffy

Hello everybody,
is it right that under optimal parameters I got only one band after IP and Western Blot? Today I did my first IP and I got 2 bands but not my band of desire
The bands were at ~ 45-50 kDa and ~ 20 kDa. Is this the destroyed fishing-ab (heavy and light chain)?
It would be very kind if someone could answer my question.
Thanks
Tiffy

[oBWhat]

yes i think they are the heavy and light chains of IgG

heavy chain IgG is around ~55kDa

light chain of IgG is around ~20-25 kDa

Although you can prevent detection of the antibody using:
* detect with protein A or G coupled to HRP instead
* cross-linking and beads
* use a biotinylated primary antibody and streptavidin HRP for detecting

It seems your problem is that you are not picking up your protein at all, not that it is hidden by the heavy/light chain bands....

what is the size of your protein? do you have a positive control (purified protein - cells that contain high amounts of the protein)?

I guess you need to optimize your IP for your protein - maybe your IP antibody is bad / not working? or your wash conditions to harsh??

[Tiffy]

Well I want to precipitate Her2 (~185kDa) and at the moment I´m using a monoclonal antibody raised in mouse. It´s isotype IgG1. As bloody beginner I used Protein A. But in the meantime I read that I should take Protein G. Could this explain my negative result. But nevertheless I don´t understand why I detected the light and heavy chain of the fishing ab. Is it that what you mean with harsh washing conditions -confusion
It is better to use different abs for IP and Western?
Tiffy

[admin]

Polyclonals are best for IPs, however I am sure some people may do it with monoclonal antibodies, although I would use a good poly if I was doing it.

Make sure you use the right immunoprecipitating agent for the antibody you pick. Protein G may only precipitate IgG isoforms so stick to that, as other protein ips may ppt other antibody isoforms... be careful

[pspn]

Hello everybody.
i m new to this forum.
would appreciate any help from the veterans here.

I am having problem with the antibody heavy chain detection from my IPs.

my protein is ~55kd.
Please give ur suggestions.

Thanks pspn

[jpap]

Hi pspn

In this case you should make sure that the primary ab you use for the western is raised in different species from the primary used in the IP, so that the western secondary will not bind the chains of the IP primary. Can you find 2 different ab against your protein?
Also run your gel long enough to get good separation around the area of 50kD, just in case there is some cross-reactivity and include a positive control so you know exactly where your peptide will migrate.

[pspn]

Thanks jpap for the reply.

that is really helpful.
will update you on this.

Have a great holiday season!