We are currently working on histone extraction from brain tissue, However, I need to know the concept of each step! from where could I get it ? or If could you provide me with it here?
For tissues (treated and untreated), weigh the sample and cut the sample into small pieces (1-2 mm3) with a scalpel or scissors. Transfer tissue pieces to a Dounce homogenizer. Add TEB buffer (PBS containing 0.5% Triton X 100, 2 mM PMSF and 0.02% NaN3) at 1 ml per 200 mg of tissue, and disaggregate tissue pieces by 50-60 strokes. Transfer homogenized mixture to a 15 ml conical tube and centrifuge at 3000 rpm for 5 minutes at 4°C. If total mixture volume is less than 2 ml, transfer mixture to a 2 ml vial and centrifuge at 10,000 rpm for 1 minute at 4°C. Remove supernatant.
Resuspend cell/tissue pellet in 3 volumes (approx. 200 µl/ 200 mg tissues) of extraction buffer (0.5N HCl + 10% glycerol) and incubate on ice for 30 minutes.
Centrifuge at 12,000 rpm for 5 minutes at 4°C and remove the supernatant fraction to a new vial.
Add 8 volumes (approx. 0.6 ml/ 200 mg tissues) of acetone and leave at –20°C overnight.
Centrifuge at 12,000 rpm for 5 minutes and air-dry the pellet. Dissolve the pellet in distilled water (30-50 µl/ 200 mg tissues).
Aliquot the extract and store the extract at –20°C or –80°C.
Answers below: in bold
cut the sample into small pieces (1-2 mm3) - increases surface area of detergent action and improves extraction.
Dounce homogenizer - It separates out multicellular systems to smaller clumps / single cell suspensions.
Add TEB buffer (PBS containing 0.5% Triton X 100, 2 mM PMSF and 0.02% NaN3) Triton - Detergent (lysis of cells) PMSF - protease inhibitor NaN3 - bacterial growth inhibitor because this is brain tissue.
Transfer homogenized mixture to a 15 ml conical tube and centrifuge at 3000 rpm for 5 minutes at 4°C - Removes lysed cytoplasm and collect the unlysed nucleii
If total mixture volume is less than 2 ml, transfer mixture to a 2 ml vial and centrifuge at 10,000 rpm for 1 minute at 4°C. Remove supernatant. - clean up step for same as above
Resuspend cell/tissue pellet in 3 volumes (approx. 200 µl/ 200 mg tissues) of extraction buffer (0.5N HCl + 10% glycerol) and incubate on ice for 30 minutes.- histones being highly basic proteins are very acid soluble and most other proteins ppt out at this step - hence throw pellet away and preserve supernatant for enriched histone fraction.
Add 8 volumes (approx. 0.6 ml/ 200 mg tissues) of acetone and leave at –20°C overnight.- Acetone outcompetes water interactions with proteins and precipitates out the histones (better in cold. slow process).
One can alternately use TCA precipitation protocol instead of acetone at this step which i find much more efficient. google for the protocol or inbox me.
Subhojit pretty much summed up everything nicely. I would just add that some protocols suggest extracting the nuclear fraction with H2SO4. Also, older protocols (from 1960s and 70s) suggest washing the acetone-precipitated pellet with "acidified" acetone first, then switching to acetone only. Dissolution of the dried pellet is done in water, because most distilled water preparations--unless deionized--are slightly acidic. Being highly basic in nature, it is easier to get the histones dissolved in slighly acidic solution; do not use Tris above pH 7. For some downstream applications, though, one needs to hike the pH (for example, if the histones are to be used in chromatin reconstitution reactions). In that case, the dissolved histones can be spiked with Tris to a pH of arounfd 7.5. For the modern rigorous "histone-enthusists", the following two protocols would be like gems:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2699528/ (from a "classical" histone leader, Jordanka Zlatanova),
http://www.nature.com/nprot/journal/v2/n6/full/nprot.2007.202.html (from a "modern" histone leader, C David Allis)
For your experimental needs, you may not need to peruse the above rigorously, though! Just thought you might benefit from the above!
Hi everyone
I have performed the whole protocol of histone extraction and I have obtained a pellet, however it is not getting dissolved in the specified volume of distilled water (50µl for 200mg brain tissue). Can anyone please tell me what could be the reason for this undissolved pellet and if possible, how to dissolve it.
(I have even try 0.01N Hcl) to dissolve the pellet but still no success.
Thankyou
Sorabh, I suggest 0.1 or 0.2M TrisCl pH8.0 for redissolving histones. but that i usually do after TCA precipitation (this protocol suggests acetone which i have found problematic at times).
Dear Subhojit Sen thank you.
The protocol i have followed also involve 100% TCA addition to supernatant. However at later steps it involves washing with acetone. Should i add just Tris Hcl buffer pH8.0 instead of water to dissolve the pellets?
Sorabh, I hope you understand the difference between "acid-extraction" (typically done with HCl or H2SO4) and "acid precipitation" (typically done with TCA or PCA) of histones. When the tissue/cell lysate is treated with HCl or H2SO4, the nucleic acids precipitate along with numerous other proteins, leaving the highly basic proteins (histones) still soluble. That soluble fraction is precipitated with acetone. This fraction is essentially DNA-free, and thus is trouble-free in dissolution in water.
On the other hand, according to conventional TCA protocols, it "precipitates" histones, but does not "extract". That means, it precipitates histones along with DNA. That nucleohistone precipitate will not go into solution unless denatured by high urea-Nacl or Guanidine hydrochloride. I suspect your protocol (or the way you did your experiment) brought DNA alongside the histones. You can "extract" histones with TCA, but the TCA concentration has to be ~5% and you must disrupt all DNA-histone contacts beforehand to be able to successfully extract histones. The histones thus extracted can be precipitated either by acetone or by TCA (now 35% v/v) and washed with acetone. Dissolve in water. Read the above two articles and follow referenced articles to develop a better understanding.
I could possibly suggest a thing or two if you provide your detailed protocol and describe what exactly you did. I cannot understand, though, how Subhojit dissolves the histones in 0.1 or 0.2M TrisCl pH8.0.
What if i increased the water needed for dissolving the pellet .. or even tried not to let it completely dry from acetone before adding water . I had found that after adding the double volume of watter ( 100microliter per 200 mg tissue ) the pellet dissolved .
and any later adjustment of the protien conc could be in water or what ?
If there is residual acetone, the pellet will be hard to dissolve. Remember the reason for dissolution: H2O molecules form extensive H-bonds with participating side-chain moieties on proteins. In presence of acetone (which is an organic solvent) hydrophilic moieties would not be exposed adequately, with non-polar surfaces being exposed. Therefore, histones will not dissolve properly if there is residual acetone. This is analogous to trying to dissolve a DNA pellet with, say, 20% ethanol.
Here is a caution, though! Over-drying makes it harder to dissolve the pellet. Under-drying or over-drying cause problems ONLY when there is substantial contamination from other proteins or DNA. If your preparation is uncontaminated, you should not face any difficulty at all.
You can dissolve the histone pellet in whatever volume of water you want. The only constraint is the histone concentration. It should not so dilute that you cannot take sufficient amount of histones in your down-stream reactions. And your experience is true: solubility is increased by increasing the solvent volume.
Later adjustment of the protein concentration should be in water. You can have 0.2mM PMSF (to prevent proteolysis), while some also recommend 15mM 2-mercaptoethanol (to keep the two H3 molecules from forming a disulfide linkage). Once the histone is all dissolved, you can spike Tris (pH 7.5-8.0) to final 10-20mM if required, but not necessary. In special cases, one may need to store histones in octameric form. That requires some special procedures.