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Methyl Binding Protein-1 (MBD1) / Immunofluorescence


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#1 SelcenC

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Posted 05 October 2011 - 09:07 PM

Hi,

I am currrently on anti-MBD1 staining optimisation in mouse embryonic fibroblasts for immunofluorescence.
I increased blocking serum concentration. And reduced primary antibody concentrations. But non-specific IgG is highly seen so far (with FITC). I fixed cells 4%PFA for 30min, then permeabilise PBS with tween-20 and triton-x.

I am wondering if anyone does specifically work on MBD1 staining for immunostaining...

Thanks and good luck to everyone !

Selcen

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#2 methylnick

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Posted 11 October 2011 - 02:29 AM

Do you have a representative image? I have done MBD2 long long ago and from memory it stains the chromosomes quite evenly with centrmere as more intense.

All comments and communication are my own personal ones, and are not tied to any of my affiliations. 


#3 SelcenC

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Posted 26 October 2011 - 06:37 PM

Hi,

I just want to let you those interested know.

Optimisation is complete for MBD1 immunostaining in mouse embryonic fibroblasts.

The protocol is as follows:

1. fix cells with 4%PFA for 30min at room temp
2. wash cells with 1XPBS (with 0.05% tween-20) for 10min X 3
3. permeabilise with PBS (including 0.5% tween-20 and 0.5% triton-x) for 40 min. at room temp
4. 4N HCl treatment (including 0.1% Triton-X)for 10 min at room temp
5. wash cells with 1XPBS (with 0.05% tween-20) for 10min X 3
6. Block cells with 30% GOAT serum at 4C overnight
7. Primary antibodies in 2%BSA/PBT (5% goat serum included in final volume) as 1:50 at 4C overnight (Important: USE rabbit IgG stock as 0.2 mg/ml)
8. wash cells with 1XPBS (with 0.05% tween-20) for 10min X 3
9. Secondary antibody incubation with FITC (1:200)in 2%BSA/PBT for 1 hour at room temp (DARK)
10. wash cells with 1XPBS (with 0.05% tween-20) for 10min X 3
11. last wash with 1xPBS
12. Mount slides

The products included in this protocol:

Rabbit IgG control: Sigma I5006
MBD1 antibody: Abcam ab3753
FITC: Sigma F1262 (anti-rabbit in goat)

Enjoy !!

:)

- Selcen -





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