doi: 10.1101/pdb.prot4939
Cold Spring Harb Protoc; Ian M. Fingerman, Hai-Ning Du and Scott D. Briggs In Vitro Histone Methyltransfera Assay Service
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In Vitro Histone Methyltransfera Assay
Ian M.Fingerman,Hai-Ning Du,and Scott D.Briggs1
Department of Biochemistry and Purdue Cancer Center,Purdue University,West Lafayette,IN47907-2064,USA
INTRODUCTION
Histone methyltransferas catalyze the addition of one or more methyl groups to a specific lysine or
arginine residue within histones.Currently,there is a great deal of interest in histone methyltrans-
feras,becau mutations and misregulation of the genes encoding the proteins have been linke
d
to various cancers and other dias.Many genes encoding putative histone methyltransferas have
been identified in eukaryotes,but the proteins they encode have not been functionally characterized.
This protocol describes an in vitro assay for histone methyltransfera activity that us bacterial cell
extracts in which expression of a methyltransfera of interest is induced.In many cas,purification
of the enzyme is unnecessary,making this experiment ideal for pilot studies.Bacterial cell extract con-
taining the methyltransfera of interest is incubated with S-adenosyl-L-[methyl-3H]-methionine and
various histone substrates,many of which are commercially available.Incorporation of the methyl-3H
can be measured easily by scintillation counting.The labeled substrate is visualized by SDS-polyacry-
lamide gel electrophoresis(PAGE)followed by fluorography.This allows the substrate specificity and
activity of a histone methyltransfera of interest to be readily characterized.
RELATED INFORMATION
元宵节月亮This protocol was adapted from Strahl et al.(2001).Protocols for histone and nucleosome core particle
isolation can be found in Schnitzler(2000).
1Corresponding author(sdbriggs@purdue.edu)
Plea cite as:CSH Protocols;2008;doi:10.1101/pdb.prot4939www.cshprotocols
METHOD
Expression of Recombinant Methyltransferas
1.In a14-mL sterile polypropylene tube,inoculate4mL of LB plus antibiotic with bacteria contain-
先进个人推荐理由
ing an expression plasmid encoding the methyltransfera of interest.
Cultures can be started from frozen glycerol stocks or from single colonies on an agar plate.
2.Incubate culture overnight with shaking at37°C.
3.Inoculate4mL of LB liquid medium containing antibiotic with0.2mL of the overnight culture,
天气热的句子measures between0.6and0.8.
and incubate with shaking at37°C until OD
600
4.Split the culture into two2-mL aliquots in new14-mL culture tubes.
5.To one tube,add IPTG to a final concentration of0.4mM.Do not add IPTG to the cond tube.
This rves as an uninduced control.
6.Incubate both cultures with shaking for4h at room temperature.
7.Transfer the cultures to microcentrifuge tubes and harvest cells by centrifugation in a microcen-
trifuge at14,000rpm for1min.
8.Remove and discard the supernatant.At this point,the collected cells can be frozen and stored
at−80°C until procesd.
探春人物形象分析
The freezing step is not esntial and the protocol can be continued if desired.
Preparation and Analysis of Bacterial Cell Extracts
9.If cells have been previously frozen,thaw them on ice.Resuspend the cells in0.2mL of bacterial
lysis buffer.
喝了咖啡能喝酒吗10.Sonicate the cells as follows,while suspending the tube in an ice water bath:
i.Using a Misonix programmable sonicator,t the program for10c total sonication time,
with a2-c pul followed by a1-c pau,and an output of level3(9W).
ii.Allow the cells to rest for2minute on ice.
11.Collect the cells by centrifugation in a microcentrifuge at14,000rpm for5min.
12.Repeat Steps10and11.
13.Transfer the supernatant to a new microcentrifuge tube;this is the soluble cell extract.The remain-
ing pellet is the insoluble fraction.
14.Add glycerol to the soluble fraction to a final concentration of10%.
Samples can be aliquoted and stored at−80°C at this point.
15.Combine a10-µL aliquot of the soluble fraction with10µL of2X SDS sample buffer.
16.Resuspend the insoluble fraction in200µL of2X SDS sample buffer.
If the insoluble sample becomes too viscous for SDS-PAGE,sonicate it using the conditions in Step10,but with-
out using an ice bath.
17.Boil all of the samples for5min prior to gel electrophoresis.
18.Analyze both soluble induced and uninduced(10µL)and insoluble induced and uninduced(10
µL)fractions by SDS-PAGE on a12%SDS polyacrylamide gel(e SDS-Polyacrylamide Gel
Electrophoresis of Proteins)followed by staining with Coomassie Blue.
19.If expresd protein is detectable in the soluble fraction by Coomassie Blue staining,assay the
sample for histone methyltransfera activity as in Steps21-39.
If the protein is not detectable in the soluble fraction after staining with Coomassie Blue,analyze the samples
by Western blotting using antibodies that detect your protein of interest.If the expresd protein can be visual-
ized by Western blot analysis,perform the histone methyltransfera assay.
See Troubleshooting.
20.Store the soluble protein extracts at−80°C.
In Vitro Histone Methyltransfera Assay
21.Set up a1.5-mL microcentrifuge tube on ice for each assay.
22.To each tube,add10µL of2X histone methyltransfera buffer.
23.Add1µL of S-adenosyl-L-[methyl-3H]-methionine.
From this point onward,reagents are radioactive and must be handled with appropriate caution.
24.Add desired histone substrates.
Substrates can be core histones,purified recombinant histones,histone peptides,or purified nucleosomes.The
amount of substrate ud in each assay varies bad on concentration.Typically,1µg of each recombinant his-
tone to be tested,1-5µg of histone peptide,4-8µg of core histones,or4-8µg of nucleosomes is sufficient.U
enough substrate to allow detection by SDS-PAGE followed by Coomassie Blue staining.If the methyltransfera
of interest is uncharacterized,test it against all of the mentioned substrates to determine specificity.
25.Add1-2µL of prepared bacterial extracts from Step14.
Using more than2µL of bacterial extract is not recommended.Set up two negative control reactions:one
using bacterial extract from uninduced cells and one without bacterial extract.Add bacterial lysis buffer in
place of extract in the latter control.Set up a positive control reaction as well using a known methyltransfera,
such as Saccharomyces cerevisiae Dot1or Set2for nucleosome substrates or human Set7/9for histone or
peptide substrates.
26.Add H
2
中考满分作文记叙文O to a final volume of20µL.
27.Incubate the reactions in a temperature-controlled shaker at1000rpm for30min at20°C.
Alternatively,carry out the reaction at room temperature with occasional mixing by hand.
28.Dilute a1M NaHCO
3(pH9.0)stock solution to make a50mM NaHCO
3
(pH9.0)working solu-
tion.Make this stop/wash solution fresh,just before u in the next step.
29.Stop the reactions as follows:
i.Spot10µL of each reaction onto Whatman P-81paper squares(2cm×2cm)which have
been labeled with a pencil and arranged on a glass plate.
ii.Immediately add2µL of5X SDS sample buffer to the remaining half of each reaction for gel analysis.
Store the samples at−20°C until Step31.Samples can be stored at−20°C indefinitely,and SDS-PAGE
analysis can be performed at your convenience.
iii.Place the filters into a1-L beaker.
iv.Add250mL of50mM NaHCO在线营销
3(pH9.0)to the filters.Place them on a room temperature
platform shaker and wash for5min.
The shaking speed should be fast enough to keep the filters suspended off the bottom of the beaker.
v.Carefully pour off the wash buffer into a radioactive waste receptacle.
vi.Repeat Steps29.iv and29.v three more times for a total of four washes.
vii.After the final wash,spread the wet filters on paper towels to dry.
30.Quantify the activity of each reaction by liquid scintillation counting.
Save the samples and count them again the next day,becau the counts will increa over the first veral hours.
See Troubleshooting.
31.For gel analysis,run the samples from Step29.ii on a15%SDS-PAGE gel(e CSH Protocols article
SDS-Polyacrylamide Gel Electrophoresis of Proteins).
For peptide substrates,do not let the dye front run off the gel;take into consideration the molecular weight of the substrates and run gels accordingly.Include in the gel prestained molecular weight protein standards in the 6-10kD range.For optimal resolution of the histone proteins run the gel as far as possible without running the samples off.
