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I'm running ChIP based off of a protocol in Nature Protocols "Fanelli et al, Vol 6. No. 12, 2011. p. 1905-1919" for ChIP of FFPE tissues. I've worked with ChIP before on cell culture, and found that I got horrible foaming unless I did many, many rounds of 10 sec pulses, 20% duty for infinity (probably more like 5 reps but it felt like infinity). This protocol combines MNase and sonication by doing Low power sonication, MNase, then high power sonication.
Well I get bad foaming on high power. I have a Branson 450 sonifier and I'm using a tapered 1/8" tip (though I have a stepped tip as well), 250µl volume in 1.5ml tube, and I can use a max output of 3 without foaming. So what is the advantage of high power sonication? Are the fragment lengths more consistant? Does it favor a larger or smaller fragment size (ie 100bp vs 1,000bp)? Is it just faster?
Like everyone else in the world, sonicating is the bane of my work life.
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non-specific staining using with Immunos
10 August 2012 - 08:29 AM
I have an antibody that I have had to troubleshoot with so much it's making me bananas. The staining itself is weak, and tends to get non-specific staining that masks true expression. It's a poly-clonal antibody, and I'm using it on FFPE sections 8µm thick using vector reagents. ALKP enzyme kits, and vector Red as my staining. I've upped the antibody to 1:25, do antigen unmasking with citrate buffer, quench endogenous ALKP with 0.2N HCl, and incubate 4˚C overnight with the primary antibody. I let the vector red incubate for 25-30 minutes, which is longer than I've ever needed to with other antibodies. I still get pink rings around my sections, or pink smears over my sections (2 jpegs below).
I'm doing something wrong, but I don't know what.
Reduce the time I incubate my primary antibody? Reduce the amount of time I leave the vector red on? something I haven't thought of?
P8100074.jpg 124.77K
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P8100075.jpg 102.37K
65 downloads
I'm doing something wrong, but I don't know what.
Reduce the time I incubate my primary antibody? Reduce the amount of time I leave the vector red on? something I haven't thought of?
P8100074.jpg 124.77K
69 downloads
P8100075.jpg 102.37K
65 downloads
