Please help me with my sonication (gel included)... - I know, everyone asks the same thing, but I'm desperate (Apr/08/2008 )
One more thing. If you really want a good looking gel, you can make your own loading buffer with less dye. As you see, the dye runs a bit above 1000 bp. In our lab we use regular 6x LB recipe with 0.25% (w/v) xylene cyanol and 0.25% (w/v) bromophenol blue. What I do is, to prepare the dye mixture but add only few drops of it to a 10xLB so that the dye is just enough to see the loading. Then you won't get the shadow on your gel
concerning the PCR, have you tried to start with more material for the IP/input or to use more material for the real-time PCR? Have you tried regular PCR? Maybe you just don't get enough material after the pulldown.
Concerning the lack of signal, maybe the crosslinking does not occur properly. You see a nice shearing but no signal. Nowadays, I have the problem that I don't get any signal in samples, but beautiful inputs. After changing everything, I suggest our formaldeyde is the enemy. We have ordered a new bottle which will be aliquoted and used only for ChIP. If that does not work, I am out of suggestions in my case.
I have been wondering about formaldehyde as well (see my recent post on this forum asking for opinions on the type of formaldehyde people use). I see something similar to you regarding the inputs. For my input, if I start with 2% of the chromatin I add to an IP sample (20ul vs 1ml), I see pretty good results with the qRT-PCR dilution series, usually with Ct values from 27-35. I'm just not getting pull-down with my antibodies.
I have been using the same bottle of 37% formaldehyde for the past three months, so maybe it is going off.
It's difficult for me to get more starting chromatin in the same volume as it means adding more embryos to the homeogenization/shearing process and I've been reluctant to fiddle with that too much, but I could try. I did try to just increase the total volume/amount of chromatin (not the concentration) and the results were unimpressive.
I think my story is similar to yours. In the beginning everything was fine with the signals and at that time I was optimizing. After a while (about half a year) the signals started to get weaker. So I thought about the sonication as you did. I tried different crosslink temperatures, new buffers, beads etc. And now (after one year), I do not get any signal at all although I am using the same crosslinking conditions as before. I don`t know what else I could try except a new bottle of formaldeyde. We use the one from Sigma (#F-8775).
Are you getting any polymerization in your formaldehyde (precipitate at the bottom of the bottle). Also, is it possible that your antibody is going bad?
If you look closely at my gel, it does seem that the smear runs the whole length of the gel. I thought this was normal and that I should focus on the brighter, mean size of the smear, but maybe that is leading me down the wrong path.
Have you performed the sonication under different conditions? Perhaps it may be handy to have some spare sonicated chromatin to use to check your samples against?
I've attached a photo of some of my results. All samples were sonicated under varying conditions. On the left was using a method that I believe wasn't lysing cells, the one on the right with a new method (different lysis buffer / time). You can see here the smear is still present along the length of the well, but is much more defined. In my opinion, your result looks more like the one on the left, and they both have 'fronts'. I've had 'bands' before right in the middle of the smear (pcr clean up fixes this, so is probably protein interference) but I think this 'band' is different to the 'front'.
I can't say whether another factor other than bad cell lysis would cause the same result, its just what it looks like to me. Does lysis buffer go off?
Actually, now that you mention it, there is a fuzzy precipitate at the bottom of my formaldehyde bottle. Not a ton of it, but a nice coating along the bottom that is clearly visible and wafts up when I swirl the bottle.
I don`t think it is a problem with the antibody. We have recently used it for Western and it has worked. In addition, I used another antibody (the protein of interest is tagged) and again no signal. This antibody is also used for Western.
It seems that old formaldehyde might be our problem??.
I have got the new bottle. I guess I`ll have results next week.
just wanna update.
We tried with a new bottle of formaldehyde and no.... No signal at all. It's so annoying.
Is the problem the beads, the antibody or what? I really don't have a clue. We will try it next week with anti-acetyl-histone3 antibody that has always been working in ChIP (quite strong signal).
How about you, kman42? Any improvement??
Well, I tried new formaldehyde to no avail. I have now switched sonicators (I'm now using an older Misonix XL) and am feeling better about things. Here's my latest gel:
Lane 1: After douncing and an initial round of sonication without SDS to release the chromatin (remember, I'm working with whole Xenopus embryos so it's slightly different than most culture protocols) and a 20 minute spin at 14k
Lanes 2-3: A sample was taken after each of two 10s pulses after SDS was added to 1%
Lane 4: After the third 10s pulse and a 20 minute spin at 14k
All samples were 10ul of a 500ul chromatin prep. I added 180ul H2O and 8ul 5M NaCl and incubated O/N at 65C. I then added 2ul of RNAse from the EZ ChIP kit and incubated 1 hr at 37C. Then I added 4ul 0.5M EDTA, 8ul 1M Tris, and 2ul Prot K and incubated 45C for 2 hours. Finally, I cleaned them up with the Upstate columns. I ran 20ul/30ul eluate, so about 1.3% of the total chromatin prep.
I'm encouraged that I am seeing a high molecular weight smear in the sample without SDS as I would expect. I'm also encouraged that the subsequent pulses seem to be making things smaller. It looks like I am losing DNA with each pulse, but maybe that it is an artifact.
Does this seem more like something I should expect? Should I just keep adding pulses until the smear gets down in size? The 8th band up in the ladder is 1kb (standard 1kb Plus ladder).
Thanks for any suggestions.