We are using EpiQuick histone extraction kit from Epigentek, but we want to know if anyone can suggest any other kit as well as any recommendation... Although this kit leads obtaining histones really fast, we are having several problems, specially when loading the samples into the gel (samples sometimes become yellow when adding the loading buffer for the acidic properties of histones). Thanks for your help!
Acid extraction of histones is very simple, you really do not need a kit. AbCam has some simple protocols for histone purification and western blotting, see:
http://www.abcam.com/protocols/histone-extraction-protocol-for-western-blot
Dear Elisabet Figuerola: Abcam sell these kits and they are very useful and easy to employ.
Here is the web link http://www.abcam.com/histone-extraction-kit-ab113476.html
Here is some info: Product nameHistone Extraction Kit
Tests1 x 100 test
Sample typeTissue, Adherent cells, Suspension cells
Assay time1h 00m
Species reactivityReacts with: Mouse, Human
Predicted to work with: all Mammals
Product overviewab113476 is suitable for a quick preparation of total histone extracts from mammalian cells and tissue samples. The minimal amount of starting materials can be as low as 105 cells or 1 mg of tissue. For the best results, the cell number should be greater than 106 cells or the tissue amount should be greater than 10 mg. A total of 100 standard extractions (use 107 cells or 100 mg of tissue per extraction) can be performed with this kit. Yield of the total histone proteins can be up to 0.4 mg per 107 cells or per 100 mg of tissue. The yield may vary depending on the cell or tissue type.
NotesShipped at +4°C. Store refrigerated at +4°C before preparation.
Tested applicationsFunctional Studiesmore details
Properties
Components
100 tests
10X Pre-Lysis Buffer
1 x 10ml
Balance Buffer
1 x 8ml
DTT Solution
1 x 20µl
Lysis Buffer
1 x 20ml
Good luck
Thank you very much for your help.
I want to know if someone who use that kit or the Abcam kit (I've read the protocol and seems the same kit from Epigentek) have samples that became yellow (that is for the acidic properties of histones) when adding the loading buffer. Is that normal?? I am adjusting the volume with Balance-DTT following the protocol and some samples turn yellow as others remain blue (and they come from the same number of cells, I don't think it is an effect of the experiment), and I ask whether these differences would lead into problems when loading them in the acrylamide gel. If someone could help with that... Thanks again.
Sample buffer turning yellow is common when doing acidic extractions. Just neutralize it with ammonia vapor (hold the tube open next to the mouth of an ammonia bottle for a few seconds until it turn blue) and continue with loading the gel.
I use the ABCAM histone extraction kit and have had a similar experience. The samples turn bright yellow after addition of loading buffer. Boiling then running the samples, however, turns the dye blue again on the 4-12% bis-tris polyacrlyamide gel.
Also, the ABCAM kit has a step involving Balance-DTT post-lysis. So I too use a solution of lysis buffer + balance-DTT to adjust volumes.
On an unrelated note, when you carry out a BCA assay, do you use the lysis buffer + balance-DTT as an additional control? If so, does it give a signal?
Hi Salmaan, thank you for your post.
Can I ask you how much time (aprox) do you boil the yellow samples till they become blue again? I tried to do that but they didn't change.
We quantify the proteins with Bradford assay, we only use the same Balance-DTT prepared in the last step of the protocol (we always prepare it fresh but I know some people who use a ready-to-use aliquot and it works too), but I don't think it is really a problem in the quantification of the samples.
Do you have problems such as blur bands in the yellow samples when you put it in a 4-12% polyacrilamide gel? Thanks again
Hey Elisabet,
I boil the samples for at least 5 minutes. After that they are still yellow in color. It is only after loading and running (stacking) that they turn blue again and you can see the dye running. That is what I meant earlier, sorry for the confusion.
I also prepare the Balance-DTT fresh but lately it has been giving me a higher signal in Bradford than some of my samples. I was wondering if you had run into a similar problem.
The gels usually run well and I do not see any "smile effects". I run into problems post-transfer, during antibody tagging and detection. I used the SuperSignal West Pico Chemiluminescent Substrate and got many blurry bands as well as unexplained artifacts, however, there were clear bands at ~17kDa (Total H3). Using a fluorescent Cy5 secondary antibody gave me cleaner results (antibody seems to be quite specific) but lower signal, making comparisons between bands more difficult.
Of course, these problems could also be associated with my gel running conditions or even errors during transfer.
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