Hi all,
I am using LiCl (500mM) wash buffer with 1% sodium deoxycholate and 1% SDS, 100mM Tris with Dynabeads.
My protocol says to use it at 4°C, but when I tried this, the buffer turns into a thick gel in which the beads cannot move towards the magnet. I know other people have had this problem on research gate and have used the buffer at room temperature and apparently the beads can move through it. I'm wondering though if anyone can explain the chemistry behind this to me and why the protocol that I was using said explicitly to store the buffer at 4°C?
Best,
Jason
The buffer gels due to the presence of 1% SDS. Solutions containing SDS are soluble in room temperatures. For 10 or 20 % SDS solutions, it is heated to 70 degrees to dissolve. For lower percentages- 1%, it dissolves at room temperature but gels at 4 degrees. I use sepharose beads for ChIP---licl wash buffer composition is same as yours except SDS.Instead it has 0.5 % NP40.
My salt wash buffers contain 0.1 % SDS and donot gels at 4 degrees. I think buffers with higher amount of SDS can be stored at room temp. We store our ChIP lysis buffer at room temp containing 1% SDS.
Hi Moumita, thank you. Could you send me your ChIP protocol please?
Dear Sir. Concerning your issue about Why does LiCl ChIP wash buffer turns into gel at 4°C. Different antibodies have different affinities for their target. Low affinity antibodies may not be able to bind the protein of interest in very stringent buffers. It is therefore worth trying different salt concentrations in the final wash with LiCl buffer (i.e. 250 and 500 mM salt).Be aware though that you want to keep the stringency as high as possible as reducing it can increase the background and affect the signal to noise ratio. Let's try washing the beads in 1 ml of ice-cold LiCl wash buffer(10mM Tris-HCl, pH 8.1, 250mM LiCl, 0.5% Triton X-100, 0.5% DOC). Invert tubes to resuspend bead pellet and incubate for 2-3 min at RT. Place tubes on magnet and allow solution to clear, ~1minute. The following below links and attached file may help you in your analysis:
http://docs.abcam.com/pdf/chromatin/A-beginners-guide-to-ChIP.pdf
http://macdougaldlab.physiology.med.umich.edu/Protocols/Chromatin%20Immunoprecipation.htm
Thanks
I'm not exactly sure why it turns into a gel but the following worked for me and does not gel. I tried making the following several times without starting from stock solutions and the resulting buffer also turned to gel. I think it's also important to add the LiCl last.
Make stock solutions as follows:
4M LiCl in H2O
1M Tris-HCl pH 7.4
10% Deoxycholate in H2O
10% NP-40 in H2O
My final concentrations are:
250mM LiCl
100mM Tris-Hcl
1% Deoxycholate
1% NP-40
Bring final volume up using H2O
Hope that helps!
I am having the same problem of the buffer turning into a gel, but not only at 4C. also at room temperature. In the summer, my buffer would take longer to turn into gel (several hours). Now it takes less than an hour for the stock buffer to solidify.
Even with freshly made buffer, the gel still forms when my strip tubes touch the magnet, which is usually cold to touch at room temp. Warming the samples at 37C did not help - and probably compromises the rest of the protocol.
Samantha Jo Mentch , did it matter to you in what sequence you added each reagent (as long as you kept the LiCl last)? My buffer protocol is the same as yours. I'd appreciate any tips. Thank you!
Danuta Sastre I have exactly the same problem as you mentioned, have you solve the problem？
Quancan Hou I unfortunately still don’t know what causes the problem. I make the buffer fresh every time to avoid unpleasant surprises. When I manage to fix it, I'll let you know. Or if you figure it out first, please let me know!
Danuta Sastre The SDS in the buffer normally precipitate at low temperatures that forms the gel. Try reducing the concentration of SDS to 0.1% and it should be fine at 4C.
I had this problem recently, I used this buffer 50 mM HEPES pH7.5, 1 mM EDTA, 0.7% sodium deoxycholate, 500 mM LiCl and protease inhibitor cocktail for CHIP washes and it became a gel even at room temp.
After reading a little bit I found out that the pH can be involved, so I tried replacing the HEPES with TRIS buffers in different pH levels, and indeed pH7 solidify faster than 7.5, and 7.5 solidify faster than pH8, but still it solidify. Then I found that other guy in the lab has the same buffer recipe but with 1% NP40 and TRIS pH8. When I tried making the same buffer but with or without 1% NP40 I found that the NP40 was the key factor. Including it in the buffer prevented the buffer solidification completely even in ice.
The problem is due to the sodium deoxycholate. I solved the problem just by changing the sodium deoxycholate from another manufacturer. Cat# 30970 (Sigma-Aldrich) works very well.