histone analysis product overview 2012

Histone AnalysisProduct 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, repressed-

H3K27me3, 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 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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• Advantages– 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


• 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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• 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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• Free software available online– Array Analyse Software can be

downloaded from website– Software only works on PCs

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• 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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• Lake Placid has tested some of our top selling antibodies - this data will be added to the CofA and website as availableTo 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

Visit www.activemotif.com/hiselisa for a current list

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

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http://www.activemotif.com/hiselisa

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

• 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 Lys36PathScan dimethyl Lys4PathScan dimethyl Lys9PathScan monomethyl Lys4PathScan pan-methyl Lys9PathScan trimethyl Lys27PathScan trimethyl Lys4PathScan Total Histone H3PathScan phospho Histone H3 Ser 10Also 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


• Available:– 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 C-terminal 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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• 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 acetyl-modifications 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 Fluor-de-

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:– 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

+ developing solutionReacts with the deacetylated substrate

Substrate : acetylated peptide+ HDAC sample

+ inhibitors

Incubate at 37˚C for 30-60 minutes

+Stop solution

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:– 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 Weakness

Arbor Assays DetectX Histone Demethylase Fluorescent Activity Kit

Includes both Jmj-type and LSD1 HDMs. However, uses H3K4me2 peptide substrate

BPS Bioscience

LSD1 Fluorescent Assay Kit Measures hydrogen peroxide

Uses a methylated peptide substrate

Works with LSD1 only

Biomol -

Enzo Life Sciences

LSD1 Fluorimetric Drug Discovery Kit Measures hydrogen peroxide

Uses a H3K4me2 peptide substrate


Ex. 530, Em. 590

Epigentek

(Kit also sold by Abnova)

EpiQuick Histone Demethylase LSD1 Activity/Inhibition Assay

Antibody-based assay that provides a fluorescent readout that is inversely proportional to HDM activity (negative assay). Both capture and detecting antibodies are used. Fluorescent settings indicate a hydrogen peroxide detection

Uses a H3K4me2 peptide substrate


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


• Active Motif offers a variety of enzymes specific for modifying histones– Acetyltransferases– Deacetylases– Methyltransferases– Demethylases– Kinases

• For a complete list visit– www.activemotif.com/hismodenz

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• 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 kitChromatin 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 assaysShows what factors are required for transcription.

•In vitro histone acetyltransferase assaysShows if associated factors are required for histone acetylation.

•In vitro ChIP assaysShows 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/CompetitorsBenefits/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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histone analysis product overview 2012

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