Transcript Histone Purification Mini Kit

Histone Analysis
Product Overview
2012
Histones
• Histones are alkaline proteins (H2A, H2B, H3
and H4) that package DNA into structural units
called nucleosomes.
• Primary protein components of chromatin. 147
bp DNA winds around octameric nucleosome
core particle consisting of dimers of each core
histone.
• Play a significant role in gene regulation
• Undergo post-translational modifications
(acetylation, methylation, phosphorylation, etc)
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Why are Histones important?
• It controls gene regulation (‘Histone Code’)
– Regulates binding/accessibility of regulatory proteins
and transcriptional machinery via:
» Chromatin compaction
» Histone modifications (ie active-H3K4me3, repressedH3K27me3, poised/bivalent-both)
• Aberrant histone regulation is associated with
improper gene expression and DNA damage
and can lead to developmental defects and
cancer
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Histone Analysis
• Histone Purification
• Histone Peptide Array
• Histone Modification ELISAs
• Recombinant Histones
• HAT
• HDAC
• HDM
• Histone modifying enzymes
• Histone antibodies
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Histone Purification Kits
Histone Purification Kit
• Customer
– Interested in obtaining pure fractions of histone
proteins while preserving post-translational
modifications
– Histones are not isolated under normal nuclear
extraction procedures. They require acid extraction
• Format
– Two kit types available:
Histone Purification Kit contains 10 rxns
Histone Purification Mini Kit contains 20 rxns
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Histone Purification Kits
• Histone Purification Kit
– Single fraction of H2A, H2B,
H3 & H4 proteins
– Or fractions of H2A/H2B
and H3/H4 proteins
• Histone Purification Mini Kit
– Single fraction of H2A, H2B,
H3 & H4 proteins
– Works from 8x106 cells or
1g of tissue
– Fast and easy
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Histone Purification Kits
• Advantages
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Preserves post-translational modifications
Fast
Works on cell and tissue samples
Allows elution of histones together in one fraction or as separate
H2A/H2B and H3/H4 fractions
– Histone purification kit provides better input sample for Histone
Modification ELISAs than acid extraction protocol
• Competitors :
– Epigentek EpiQuik (or Abcam EpiSeeker) Total Histone Extraction
Kit – 100 rxn format, only extracts total histone fraction.
– This method is patented, so we have an advantage with our
exclusive kit!
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MODified™ Histone Peptide Array
MODified™ Histone Peptide Array
• 384 Different histone modification combinations
• 59 post-translational modifications for acetylation,
phosphorylation, methylation and citrullination
• Peptides are 19mer long with up to four separate
modifications on each peptide
• Excel sheet on website as reference for spots and their
location on array
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MODified™ Histone Peptide Array
• Customers
– Characterizing antibody cross-reactivity
– Screening for protein or enzyme interactions with histone modifications
• Format
– Single array
– 5 pack array
• MODified Array Labeling Kit available
– Contains buffers, c-Myc control antibody, rabbit and mouse HRP-conjugated
secondary antibodies and ECL reagents for chemiluminescent detection of
MODified Peptide Array
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MODified™ Histone Peptide Array
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MODified™ Histone Peptide Array
• Free software available online
– Array Analyse Software can be
downloaded from website
– Software only works on PCs
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MODified™ Histone Peptide Array
• Advantages
– Histone specific panel testing H2A, H2B, H3 & H4
– Up to 4 modifications per 19mer peptide enables analysis of the
effects of neighboring modifications on site recognition and
binding
– Detects like a Western blot (Colorimetric or Chemiluminescent)
– Works with antibodies, proteins or enzymes
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MODified™ Histone Peptide Array
• Lake Placid has tested some of our top selling antibodies - this data
will be added to the CofA and website as available
To see this data go to www.activemotif.com/arraytested
• Array has been validated for use with histone modifying enzymes,
such as HMT (data available in product manual)
• Data for 39161 Histone H3 trimethyl Lys9 pAb is shown below
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Histone ELISA kits
Histone Modification ELISAs
Current available Histone ELISA kits :
• Total Histone H3 ELISA
• Histone H3 mono-, di- and tri-methylated Lys4 (H3K4) ELISAs
• Histone H3 di- and tri-methylated Lys9 (H3K9) ELISAs
• Histone H3 mono- and tri-methylated Lys27 (H3K27) ELISAs
• Histone H3 acetyl Lys9 (H3K9) ELISA
• Histone H3 acetyl Lys14 (H3K14) ELISA
• Histone H3 phospho Ser10 (H3 pS10) ELISA
• Histone H3 phospho Ser28 (H3 pS28) ELISA
Visit www.activemotif.com/hiselisa for a current list
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Histone Modification ELISA Flow Chart
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Who are our customers?
-Researchers involved in epigenetics and histone modifications
-customers wanting to quantitate methylation of lysine residues within their
samples.
-Customers currently using our histone purification kits (Catalog Nos. 40025
& 40026) as well as our modification specific histone antibodies may be
interested in this product.
Questions to ask :
- Are you currently studying histone modifications such as lysine
methylation?
- Are you currently performing immunoblotting experiments to do this
analysis?
- Are you trying to quantitate the amount of histone methylation in
your samples?
- Are you trying to normalize for total histone H3 levels?
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Histone ELISA kits : benefits
• Modification specific antibodies that do not cross-react with other
site- or degree-specific methylation
• Recombinant protein control (positive control included) for
quantification with site- and degree-specific lysine methylation
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Optimized buffers and protocol
• Lower price than other kits on the market
• Multiple modifications to chose from
• Can compare dimethyl Lys9 with trimethyl Lys9, or normalize
against Total Histone H3. Additional modifications anticipated for
release this year.
• Less than 4 hours
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Histone ELISA kits : competitors
Company
Product
Weakness
Cell Signaling
PathScan
dimethyl
Lys36
PathScan
dimethyl
Lys4
PathScan
dimethyl
Lys9
PathScan
monomethyl
Lys4
PathScan
pan-methyl
Lys9
PathScan
trimethyl
Lys27
PathScan
trimethyl
Lys4
PathScan Total Histone H3
PathScan phospho Histone H3 Ser 10
Also has acetyl modification kits
No positive control, assay takes 4 hrs to overnight to
complete
Invitrogen
(Biosource assay)
Histone H3 (Total)
Histone H3 phospho Ser10
Limited product offering
Bethyl Labs
Histone H3 PathProfiler Kit (either
Total H3 or Phospho Ser10)
Limited product offering, no positive control
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Recombinant Histone Proteins
Recombinant Histone Proteins
• Available:
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Recombinant unmodified histones
Recombinant methylated histones
Recombinant acetylated histones
Recombinant phosphorylated histones
Biotinylated histones for use in FRET assays
• Applications:
– Can use these as substrates in functional assays, or
in chromatin assembly kit to generate their own
chromatin
– Good substrates for Pharma experiments
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Generation of Recombinant Histones
• Prepared using one of two patented technologies:
– Expressed Protein Ligation (EPL)
» A semisynthetic method in which an expressed protein containing Cterminal thioester is ligated to a peptide containing an N-terminal cysteine
via a chemoselective reaction.
» Preserves the native amine bond and natural protein structure, more
closely mimics natural histones.
– Methylated Lysine Analog (MLA)
» A chemical alkylation reaction that substitutes aminoethylcysteine, a
lysine analog that is structurally and chemically similar to lysine, for the
existing lysine at the desired residue.
» Provides precise control over the site and degree of methylation. Despite
the substitution, recombinant proteins show functional similarity to native
proteins.
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Recombinant Histone Proteins
• Dot blot was used to confirm
the identity of H3K79me2
recombinant protein
• 1 µg of various recombinant
proteins were spotted onto a
PVDF membrane
• Histone H3 dimethyl Lys79 pAb
(Cat # 39143) was used at a
1:1,000 dilution
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HAT (Histone Acetyltransferase) Assay
What is HAT?
• Histone AcetyTransferases are enzymes that put acetylmodifications on histone tails.
Ex: p300 and PCAF
• Role in gene regulation.
• Involved in cancers and disease and are the targets of new drug
development.
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HAT Assay Kit
• Customers
– Academic researcher studying Chromatin biology
– Pharma companies studying histone acetyltransferase inhibitors or
screening inhibitors as potential drug compounds
• Format
– 96 reactions
– Fluorescent assay (ex. 360-390nm, em. 460-490nm)
– Includes enough p300 catalytic domain protein to screen
96 wells of inhibitor compounds
– Includes anacardic acid as a control inhibitor
– H3 and H4 N-terminal peptides are included as HAT substrates
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Protocol
• Used for testing activity of a purified protein and inhibitor
compounds.
• In a 96 well plate, add protein, (inhibitors),substrate,
Acetyl-CoA, buffers, incubate 10 to 30 minutes at room
temp, develop and read on plate reader.
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HAT Assay Kit
– Quick and sensitive method to screen for HAT activity
or inhibition
– Quantitative method when using standard curve
– Can assay both H3 and H4 acetyltransferase activity
– Includes control p300 protein and an inhibitor
– This kit has not been optimized for cell lysates,
however, it is possible (we did not test but it’s
published) if the extracts are treated to remove free
sulfhydryl groups in the sample.
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Who would buy the HAT assay kit?
• Someone who is trying to target HAT activity in a
disease pathway and needs to test treatments.
• Someone who has isolated a new protein that may be
another Histone Acetyltransferase and needs to test
activity.
• Someone who is working with HDACs may also need to
test for HAT activity.
Why?
- To screen inhibitors of HAT activity
- To test activity of a new purified HAT
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Competitors and Sales Strategy
• Primary competitors:
– Upstate (Millipore), but this is a sandwich ELISA format
– Formats like ours: BioVision, Cayman Chemical, MBL international.
• Advantages:
– Pricing, ours is cheaper. Our kit is very similar to the Cayman kit. They
advertises theirs as an “inhibitor screening” assay; but, we tested it
and it does not work to test inhibitor activity.
• Sales strategy:
– Kit is similar to other kits already on the market such as BioMol Fluorde-Lys assay
– Cross sell with recombinant proteins:
» Recombinant p300, catalytic domain
» Recombinant GCN5
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HDAC Assay Kit
What are HDACs?
• Histone Deacetylase enzymes remove acetyl groups
from histone tails.
• Who studies HDACs? Academics, cancer researchers.
HDACs are the target of many new screenings for drug
compounds.
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What is this kit?
• Colorimetric and fluorescent HDAC assays available.
• Used to test HDAC activity in nuclear extracts.
• 96-well plate format, HeLa nuclear extract included as a
control.
• Kit contains all necessary buffers and substrates:
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HeLa nuclear extract
Trichostatin A (HDAC activity inhibitor)
Acetyl-lysine substrate (colorimetric or fluorometric)
96-well assay plate
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HDAC Assay Method
Substrate : acetylated peptide
+ HDAC sample
+ inhibitors
Incubate at 37˚C for 30-60 minutes
+Stop solution
+ developing solution
Reacts with the deacetylated substrate
chromophore (color kit)
or fluorophore (fluor kit)
Measure the signal
intensity
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HDAC Assay Kits
• Advantages
– Works with either nuclear extracted samples, purified
recombinant HDAC enzymes or enzymes isolated by
immunoprecipitation
– Available in either fluorescent or colorimetric formats
– Quantitative method when using the standard curve
– Includes a control extract and inhibitor
• Can bundle with HAT assay, nuclear extract kit, HDAC
antibody customers
• Customers
– Chromatin biology
– Pharma companies studying HDAC inhibitors
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Histone Demethylase Assay Kit
What are HDMs?
• Histone Demethylase enzymes remove methyl groups from
histone tails.
• LSD1 and Jmj; LSD1 specifically demethylates H3K4, an
important mark associated with transcriptional activation.
• Who studies HDMs? Academics, cancer researchers. LSD1
is important target for drug discovery.
• The Histone Demethylase Assay is used to screen activity of
purified or recombinant lysine-specific demethylase (LSD1)
enzymes.
• Customers
– Academic researcher studying Chromatin biology
– Pharma companies studying histone demethylase inhibitors or
screening inhibitors as potential drug compounds
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What is this kit?
• Fluorometric 96-well plate format.
• Assays activity/inhibition of purified or recombinant
LSD1 enzyme. Can be used to analyze overall histone
conversion efficiency of dimethyl substrate to either
mono- or un-methylated state.
• Kit contains all necessary buffers and substrates:
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Control LSD1 enzyme
Recombinant Histone H3K4me2 substrate
Demethylation standard for quantification
96-well black half area assay plate
• How does it work?
– LSD1 demethylation of the H3K4me2 substrate causes release
of formaldehyde at each step, which reacts with the Detection
reagent and generates a fluorescent signal
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HDM Assay Protocol
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HDM Assay kit : competitors
Company
Product
Arbor Assays
DetectX
Histone
Fluorescent Activity Kit
Weakness
Demethylase
LSD1 Fluorescent Assay Kit
Includes both Jmj-type and LSD1 HDMs. However,
uses H3K4me2 peptide substrate
Measures hydrogen peroxide
BPS Bioscience
Uses a methylated peptide substrate
LSD1 Fluorimetric Drug Discovery Kit
Works with LSD1 only
Measures hydrogen peroxide
Biomol -
Uses a H3K4me2 peptide substrate
Enzo Life Sciences
Works with LSD1 only
Ex. 530, Em. 590
EpiQuick Histone Demethylase LSD1 Antibody-based assay that provides a fluorescent
Activity/Inhibition Assay
readout that is inversely proportional to HDM activity
Epigentek
(negative assay). Both capture and detecting
antibodies are used. Fluorescent settings indicate a
hydrogen peroxide detection
(Kit also sold by
Abnova)
Uses a H3K4me2 peptide substrate
Works with LSD1 only
Ex. 530, Em. 590
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HDM Assay Kits
• Advantages
– The formaldehyde excitation/emission spectra allows for use of
common filter sets (unlike hydrogen peroxide byproducts Ex
550/Em 590)
– Only company to use a recombinant H3K4me2 substrate, mimics
in vivo conditions better than peptide substrates.
– Includes positive control LSD1 and standard for quantification
– Fast, 2.5 hour protocol
• Cross-sell products include:
– LSD1 enzyme, recomb. Methylated Histones, Histone ELISAs,
Histone Purification Kits, Methylated Histone antibodies
• Customers
– Chromatin biology, cancer, transcription biology, DNA repair
– Pharma companies studying HDAC inhibitors
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Histone Modifying Enzymes
Histone Modifying Enzymes
• Active Motif offers a variety of enzymes specific
for modifying histones
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Acetyltransferases
Deacetylases
Methyltransferases
Demethylases
Kinases
• For a complete list visit
– www.activemotif.com/hismodenz
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Histone Modifying Enzymes
• MODified Histone Peptide Array
tested with histone
methyltransferase G9a
• Top: 25 µM G9a (Cat. # 31327)
and 1 mM AdoMet
• Bottom: 25 µM G9a mutant (Cat.
# 31328) and 1 mM AdoMet
• The wild-type G9a protein was
able to establish new methylation
sites on the MODified Histone
Peptide Array, as detected with
the Histone H3 dimethyl Lys9
antibody
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Chromatin assembly kit
Chromatin assembly kit : overview of the kit
• What is it?
– This kit enables customers to assemble chromatin on linear or
circular input DNA in vitro to be used for downstream assays (ie
in vitro transcription and ChIP, histone mod assays.)
– This kit is like a sample-preparation kit, this kit is not the
experimental end-point.
• Why use the kit?
– To investigate the effect of the chromatin configuration on
nuclear function. Naked DNA just doesn’t always reveal the
underlying mechanisms involved in gene regulation since it does
not resemble the in vivo chromatin-like environment.
• How does it work?
– The kit contains recombinant histone chaperone and chromatin
assembly factor proteins, core histones, and ATP to assemble
nucleosomes in an ordered array on the DNA sequence of
interest.
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Chromatin assembly kit : principle
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Chromatin assembly kit : Applications
•In vitro transcription assays
Shows what factors are required for transcription.
•In vitro histone acetyltransferase assays
Shows if associated factors are required for histone
acetylation.
•In vitro ChIP assays
Shows what additional proteins associate with targeted
factors and sequences. Could just be a control to show the
in vitro chromatin resembles in vivo results.
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Benefits/Competitors
• Results in an extended ordered array of nucleosomes.
• Saves researchers the time and headache of expressing and
purifying the proteins for the assembly process themselves.
• Works with either linear or supercoiled DNA.
• Easy protocol: just mix together and incubate.
• Siliconized tubes and reagents to confirm the reaction worked
are provided.
• Competitor: Vaxron, Millipore
– Their kit is more expensive.
– This kit does not result in an ordered array of nucleosomes.
– Theirs also works with either linear or circular DNA (or PCR
products).
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Who are our customers?
• Researchers purifying histones
• Researchers working on chromatin structure
 cancer research
 transcription and influence of the chromatin on
transcription
 all researchers interested in epigenetics (cancer,
developmental diseases, transcription, chromatin
biology,retrovirology,…)
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Antibodies
Histone Antibodies
• 170 antibodies
against histones
and specific
histone
modifications
• 155 antibodies
against
chromatin
modifiers (HATs,
HDACs, HDMs,
etc)
• More added
every month
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