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Covaris, Covaris, Covaris

November 30, 2011

Like just about everything my Covaris ChIP protocol seems to work much better for some epitopes than others. My guess is that this primarily has to do with the degree of crosslinking. So I decided to increase the crosslinking time to 10 minutes like I have done with ChIP in the past and look at how well the DNA shears. There is one other little issue, stock 37% formaldehyde contains about 15% methanol. Hamid at Covaris has seen that methanol in the crosslinking reactions increases crosslinking efficiency.

In the below experiment, I took 15 cm plates of HeLa cells and cross-linked them for 10 min with 1% formaldehyde, prepared the chromatin according to my Covaris shearing protocol and sheared the DNA for 10 minutes, 20 minutes and 30 minutes (actually it was the same sample with aliquots taken every 10 minutes). In the first three lanes the crosslinking was performed with ‘fresh formaldehyde’ (formaldehyde from single use ampules) in culture media. Since the cells were taken directly from the incubator, the culture media was at 37˚C when the formaldehyde was added, although it cools over the 10-minute incubation time, which was performed at room temperature. The second set of three lanes is the same but I used an approximately three-month-old 37% formaldehyde stock solution. In the third set of three lanes the crosslinking was performed in my version of ‘Covaris Fixing Buffer’ (50 mM Hepes, pH 7.5, 0.1 M NaCl, 1 mM EDTA, 0.5 mM EGTA) according to my protocol using fresh formaldehyde. The final set of three lanes is the same as the previous three lanes except I added methanol to a final concentration of 0.4%.

From this experiment, it appears that there is not much of a difference between using fresh formaldehyde or an older 37% stock solution of formaldehyde when it comes to shearing efficiency. It also does not appear that methanol in the crosslinking reaction effects shearing efficiency in the conditions I am using. It also appears that the degree of crosslinking in media is much more than when performed in ‘Fixing Buffer’. I don’t know if this is due to the temperature or the composition of the buffers or a combination of both. I would like to note that at the 10-minute time point in the samples fixed with ‘Fixing Buffer’ there was substantial foaming in the samples, which I removed before continuing to shear. I think this is why there was poor shearing in these samples at 10 minutes, but this is just a guess.

The two western blots below show a long (upper) and short (lower) exposure of a 50 kD protein (I did a western blot of for a 120 kD protein and it looked the same). Crosslinking in media shows a huge loss in epitope as compared to ‘Fixing Buffer’. This is most likely due to increased levels of crosslinking. In all the samples there is substantial loss of epitope over the shearing time course. In previous experiments I have shown that there is very little epitope loss in the first 10 minutes of shearing. The sample fixed in ‘Fixing Buffer’ with 0.4% methanol shows less loss of epitope, although I would like to see if this is reproducible.

On a final note, perhaps I will post more on this later, crosslinking for 45 minutes in 2 mM DSG (disuccinimidyl glutarate) prior to crosslinking for 5 minutes in formaldehyde does not seem to effect shearing efficiency, so if you have a protein that is not directly bound to the DNA this may be a good way to go.

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7 Comments
  1. great post!

  2. gellybear permalink

    Hi Ethan,

    Thanks for this post! Could you also post what Covaris shearing settings you used for this experiment? (i.e. duty cycle, intensity, volume and tube, number of cells) I have been having shearing problems using the covaris – for a particular cell type I’m working with, I can’t seem to shear it down to the right size using their recommended protocol (same as the one you have in your protocol here: https://ethanomics.files.wordpress.com/2011/10/chip_covaris.pdf). Even when I increase to 25 cycles (i.e. 25 mins of shearing time), I still see a very large amount of DNA at >1kbp range. Also I always get two peaks when I run the gel…

    So it would be great to see what settings you have used here to get the right size. I also use a specific shearing buffer, rather than media. It makes the shearing process more “uniform” – since you may grow diff cells in diff media, and each media type can contain factors/proteins/chemicals that affect the rate of x-linking differently.

    • gellybear permalink

      Sorry, I meant “specific *fixing* buffer”, not shearing buffer.

      Which leads me also to ask, are you using a non-ionic buffer, or an SDS based buffer for shearing?

      Thanks again!

  3. ethanomics permalink

    I’ve sheared HeLa, MEFs and mouse ES cells and they all seem to shear with the same kinetics. With HeLa’s and MEFs I generally use one 15 cm plate/ml of shearing buffer. ES cells we use less cells but the shearing kinetics seems the same. The conditions are in my protocol which is currently here:
    https://ethanomics.files.wordpress.com/2011/10/chip_covaris.pdf
    but if it moves check the protocol tab at the top of the page. The only thing that is different is I’ve been using an intensity of 4, whereas in the protocol it says use 3. An intensity of 3 seems to take just a little bit longer to shear the chromatin and being the impatient person I am, I’ve upped it to 4.

    All the buffers are exactly as written in the protocol. The only thing I can think is are you using the TC12x12 tubes with the AFA fiber? If you do it in other tubes (apart from Covaris microTubes with AFA fibers) it does not work.

    • gellybear permalink

      I’m already using their microtube, so that’s not it. But thanks for the information you posted!

  4. Jason permalink

    dumb question but how did you run the gel above to test the effects of fixing on the epitope? Is it a native gel or is this an IP.

    • ethanomics permalink

      Sorry for the delay on this question. I’ve been a little too busy lately. It’s just a regular SDS-PAGE gel. When you reverse X-link, make an aliquot of 10 μl of chromatin. Incubate at 65˚C for > 4 hrs. Add SDS loading dye and run on a regular SDS-PAGE gel. It’s that simple.

      It’s more of a test of the sonication on the epitope, since the X-linking is reversed. So if the epitope was destroyed by X-linking it will come back after reversing the X-linking.

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