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PBS not ChIP Buffer


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#1 Happyzen

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Posted 02 April 2009 - 07:28 PM

So every so often I do sthg really incredibly stupid. And it was about time I did again!

So I was doing a ChIP using the upstate kit and instead of adding my cells to ChIP dilution buffer inthe kit (0.01% SDS, 1.1% Triton X EDTA and Tris)
I diluted cells in PBS (plus protease inhibitors) and added my antibody and rotated overnight.

I only realised my idiocy this morning.

To all the experts (it's my first one) do you think it's worth continuing with the washes?

Thanks so much for your time and expertise.

sincerly

Zena

#2 KPDE

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Posted 03 April 2009 - 11:29 AM

So every so often I do sthg really incredibly stupid. And it was about time I did again!

So I was doing a ChIP using the upstate kit and instead of adding my cells to ChIP dilution buffer inthe kit (0.01% SDS, 1.1% Triton X EDTA and Tris)
I diluted cells in PBS (plus protease inhibitors) and added my antibody and rotated overnight.

I only realised my idiocy this morning.

To all the experts (it's my first one) do you think it's worth continuing with the washes?

Thanks so much for your time and expertise.

sincerly

Zena


Sorry to say it but my guess is that you are out of luck. The detergent in the dilution buffer is important in keeping proteins from aggregating and the chromatin from sticking to everything non-specifically (including your protein A beads and the sides of the tube). My guess is that, at best you would have a seriously high background. Still, all you have to waste if you keep going is some time and some PCR master mix to analyze the results.

#3 Happyzen

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Posted 04 April 2009 - 06:07 AM

.. sad. I was afraid someone would say that.

Well, I guess I have to do the PCR monday to find out.

I'm counting on PBS not doing anything .. but yes, detergent prevents aggregation and all i can imagine is my DNA jailed in the aggregates.

.. sad. I'm going to have to grow new cells and everything.

SO STUPID!

#4 KPDE

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Posted 04 April 2009 - 11:25 AM

SO STUPID!


I'm fairly certain you're not the first person to use the wrong buffer in an assay.

#5 Happyzen

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Posted 07 April 2009 - 11:30 PM

SO STUPID!


I'm fairly certain you're not the first person to use the wrong buffer in an assay.



Well, I contiunued with the washes and performed the qPCR.

Would you believe it worked?
It did.

I included an old acetylation ChIP i'd done and it's input as a positive control and I have the exact same control in this recent ChIP .. and they are both almost identical in value
.. umm I used it again a positive control gene (gene I expect acetylation for) and negative gene (no acetylation) and the results are exactly as expected .. about 2 when I plot IP/input.
And the no antibody was negative .. i.e ip/input = 0.01 ..
I can only assume that the ChIP has worked. Any controls/experiments I can further do?

Anyway, thanks for your help.

#6 KPDE

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Posted 08 April 2009 - 01:17 PM

SO STUPID!


I'm fairly certain you're not the first person to use the wrong buffer in an assay.



Well, I contiunued with the washes and performed the qPCR.

Would you believe it worked?
It did.

I included an old acetylation ChIP i'd done and it's input as a positive control and I have the exact same control in this recent ChIP .. and they are both almost identical in value
.. umm I used it again a positive control gene (gene I expect acetylation for) and negative gene (no acetylation) and the results are exactly as expected .. about 2 when I plot IP/input.
And the no antibody was negative .. i.e ip/input = 0.01 ..
I can only assume that the ChIP has worked. Any controls/experiments I can further do?

Anyway, thanks for your help.


Well I'm very happy that it worked out. Also, very interesting that it worked. I have some tweaking to do with the buffers we use to see what is and is not necessary.

#7 Happyzen

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Posted 10 April 2009 - 12:34 AM

Well I'm very happy that it worked out. Also, very interesting that it worked. I have some tweaking to do with the buffers we use to see what is and is not necessary.



Thanks :-) i'm also very happy.
Interesting. I suppose I was using the anti-Histone (H3) aceytl antibody .. and I had about 2 million or so cells to start .. and protein A beads (with salmon sperm) The Ab is a very good Ab especially against Protein A beads.

heheh are you a PhD student? My supervisor always gets the PhD student to do stuff like that :-)
Lucky for 2 they got a nature out of it!

#8 KPDE

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Posted 10 April 2009 - 05:14 PM

heheh are you a PhD student? My supervisor always gets the PhD student to do stuff like that :-)
Lucky for 2 they got a nature out of it!


I am actually a student (graduating next month) but I'm interested in optimizing ChIP because our lab has published a couple of higher-throughput and plate based ChIP protocols. We're always looking to simplify the assay as much as possible. While I agree that optimization is scientifically uninteresting, the protocols we developed have helped the science in my projects quite a bit.

#9 Happyzen

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Posted 13 April 2009 - 04:49 PM

heheh are you a PhD student? My supervisor always gets the PhD student to do stuff like that :-)
Lucky for 2 they got a nature out of it!


I am actually a student (graduating next month) but I'm interested in optimizing ChIP because our lab has published a couple of higher-throughput and plate based ChIP protocols. We're always looking to simplify the assay as much as possible. While I agree that optimization is scientifically uninteresting, the protocols we developed have helped the science in my projects quite a bit.


Wow .. cool .. my lab has published on high Thru'put methylation analysis .. Coolen M et all and My supervisor Sue Clark helped pioneered Bisulphite conversion ..
Have you tried the millipore magna chip kit? What do you think?

At the moment, we use upstate ..




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