9 replies to this topic

[trangtrang]

Dear gals and guys,

When i used the IgG as the negative control in IF, I have a strong staining.
But the staining of primary antibody (rabbit) is quite specific. The staining of secondary antibody alone is quite dark.

I don't understand.

Please help!!!!

Thanks in advance.

[Tabaluga]

Can you give more details on your protocol (blocking etc.) ?
Have you titrated the IgG ? Take care that the concentration should be equal to your sample antibody, not necessarily the dilution.
Which company is your IgG from ? I noticed that for example the cheap IgG's from santa cruz give higher background than IgGs of better quality.
Also, do you need to use an IgG control ? It's standard for flow cytometry etc, but I think that for IF it's more common to use a secondary AB only control ?

[trangtrang]

my protocol is:
- fixation: PFA 4%
- blocking buffer: saponin/BSA/FBS; 1h/Room temparature
- primary antibody: polyclonal rabbit or IgG; OVN/4°C
- secondary antibody: rabbit; 1h/room temparature
- mounting with Prolong gold antifade.

actually, I took the IgG from other in my lab. She used this IgG for negative control in CoIP. I tried this IgG 'cause I just want to check my staining of primary antibody is specific or not.
IgG is from R&D Systems.

[mdfenko]

what is the source of the igg? if it's from rabbit then your secondary is specific for it. if it's from another species then your secondary is cross reacting to a degree.

the secondary is made against total igg from the target species.

[trangtrang]

Yes it is normal rabbit IgG .
sorry, for my bad english. But I don't understand what you means. Can you explain by different way, please?

Thanks.

[trangtrang]

http://www.rndsystem...ducts/ab-105-c/
Here the ref of this IgG.
Honestly, I dont know why they use this word "normal".

[Tabaluga]

"Normal rabbit IgG" is just the name for purified IgG antibodies from rabbit that are not targeted against a specific protein, so can be used as controls.
Your protocol looks OK. Your secondary is something-against-rabbit, I presume ?

[mdfenko]

since your secondary is against rabbit igg, using rabbit igg as a control will be a positive control not a negative control. you should expect a strong response.

secondary antibodies are against a class of proteins (ie: igg) not specific to any single protein (like a primary).

[Tabaluga]

mdfenko, on 25 February 2013 - 07:24 AM, said:

since your secondary is against rabbit igg, using rabbit igg as a control will be a positive control not a negative control. you should expect a strong response.

But only if you would expect the rabbit IgG to bind to the cells... I'd say it's a negative control because you don't expect the (unspecific) rabbit IgG to bind to the cells, unlike your specific sample antibody which binds to a certain target on the cells.

[mdfenko]

oops, for some reason (that escapes me) i was replying based on western blots.

however, it is still possible that the normal rabbit igg contains antibodies to other proteins in the cell. you could check it with a western blot.

or, better yet, try another lot of normal rabbit igg.

Edited by mdfenko, 26 February 2013 - 05:11 AM.