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Immunofluorescence - blocking


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#1 katcvet

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Posted 21 May 2011 - 01:13 AM

I have a cell line which is engineered to express a specific protein. The expression vector has C-terminal c-myc tag to allow for detection of protein expression using anti-myc Ab.

To check for protein expression I grow my cells on coverslips to 80% confluency, fix in PFA then incubate in saponin buffer (0.1% w/v saponin, 10% (v/v) FBS, in PBS) for at least an hour. Then incubate in the primary antibody mouse anti-myc (diluted 1: 50 in saponin buffer) for one hour at 4°C, then for 15 minutes in saponin buffer. My secondary antibody is goat anti-mouse Alexofluor(diluted 1: 300 in saponin buffer) incubated for one hour at 4°C in dark. I then wash my coverslips in 10% FCS in PBS for 15 minutes, and mount using Vectashield with DAPI.

I have a few questions (probably very simple and illustrating a lack of understanding!)as I've never done any immunofluorescence before and the protocol is from someone else.

The protocol seems to work in that the engineered cells express myc, the non-engineered cells don't and there's no IF I use a negative control primary Ab. What I'm unsure about is the buffers I am using - presumably the point of the first incubation in saponin buffer is to block any non-specific Ab binding, but the buffer contains bovine serum whereas my Ab are either mouse or goat - so is it ok to use bovine serum? Is the purpose of the wash in saponin buffer after incubation in primary Ab just to physically remove Ab, or does this have a blocking role too? Likewise the final wash in 10% FCS in PBS (why use this rather than saponin).

Any help much appreciated!


#2 Christo K

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Posted 21 May 2011 - 04:06 AM

Hello,

There is no problem using FBS, you can use BSA too if you want too- not too much difference. These are wildly used for blocking the samples no matter if your Ab are mouse or goat.

Yes it is better to wash after your primary Ab and after your secondary Ab before adding DAPI and mounting. You usually wash with the buffer you have diluted your Abs..

as about "final wash in 10% FCS in PBS (why use this rather than saponin)" I can't help- If it works and you have good images- use it - if not change :D.


just a last tip, I don't know if you already do this but I suggest to have a second negative control where you have transfectet cells(as the +control) but you haven’t add primary antibody, because sometimes you might have non specific binding of your secondary and much noise.

Good luck

#3 bob1

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Posted 21 May 2011 - 04:37 PM

A typical IF protocol will go something like this:

1.Fix in 2-4% PFA
2.wash lots in PBS
3.permeabilise in 0.1% tween 20 in PBS (PBS-T)
4.block in 1-10% BSA in PBS-T
5.Primary diluted in blocking solution for however long it takes
6.wash 3-5 times in PBS or PBS-T or blocking solution
7.secondary diluted in blocking solution (for alexafluors I use 1:1000-1:2000) for 1 hour.
8.wash as above
9.DAPI, 10 min in PBS-T
10. 2-4 washes in PBS
11.mount

your protocol should be OK, but I would suggest that you can probably dilute the antibodies a bit more, and wash a bit more to make sure that the antibody binding is specific.




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