PBS not ChIP Buffer - I used PBS not ChiP dilution buffer. (Apr/02/2009 )
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
Happyzen on Apr 2 2009, 08:28 PM said:
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.
.. 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!
Happyzen on Apr 4 2009, 07:07 AM said:
I'm fairly certain you're not the first person to use the wrong buffer in an assay.
KPDE on Apr 4 2009, 12:25 PM said:
Happyzen on Apr 4 2009, 07:07 AM said:
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.
Happyzen on Apr 8 2009, 12:30 AM said:
KPDE on Apr 4 2009, 12:25 PM said:
Happyzen on Apr 4 2009, 07:07 AM said:
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.
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.