thermo scientific halt protease and phosphatase...

Thermo Scientific Halt Protease andPhosphatase Inhibitor CocktailsIncluding the combination protease and phosphatase inhibitor cocktails

Product

B-PER® ReagentPI-78243, 165 mlPI-78248, 500 mlPI-90084, 50 mlPI-78260, 250 ml (2X)PI-78266, 500 ml (PBS)

B-PER Kits with EnzymesPI-90078, 250 mlPI-90079, 500 mlPI-90080, 50 mlPI-90084, 250 ml

Y-PER® ReagentPI-75768, 25 mlPI-78991, 200 mlPI-78990, 500 ml

Y-PER Plus ReagentPI-78998, 25 mlPI-78999, 500 ml

P-PER® KitPI-89803

I-PER® ReagentPI-89802, 250 ml

M-PER® ReagentPI-78503, 25 mlPI-78501, 250 mlPI-78505, 1 L

T-PER® ReagentPI-78510, 500 ml

NE-PER® ReagentPI-78833PI-78835

Mem-PER® ReagentPI-89826

Mitochondria Isolation KitPI-89874

For TissuePI-89801

Organelle Enrichment KitsPI-89839 (Lysosome)PI-89840 (Peroxisome)PI-89841 (Nuclei)

Compatibility

Reporter assays, IPs1, Western blot, GST- and histidine-tag purification

IPs1, Western blot, β-Gal enzyme assays, IEF after dialysis, GST- and histidine-tag purification

GST- and histidine-tag purification, Western Blot

1D and 2D gel electrophoresis, Western blotting, IPs1, affinity purification, activity assays

6xHis protein metal chelate chromatography, ion exchange chromatography, Western blot

Luciferase, β-Gal (low signal), CAT, kinase assays, ELISAs, immobilized glutathione, Western blot

Luciferase, β-Gal, CAT, kinase assays, Western blot, ELISAs, immobilized glutathione

EMSA (if using < 3 μl or 10%, otherwise dialyze first inThermo Scientific Slide-A-Lyzer MINI Dialysis Units),Western blot, reporter assays, IEF (after dialysis to reducesalt concentration), 2D3

Western blot and 2D3

Western blot, 2D3 Western blots, electrophoresis

Western blotting, 2D/MS, electron microscopy; disease profiling, gene expression, signal transduction and interac-tion or localization studies

Organisms/Samples

Gram(-) bacteria, S. aureus, H. pylori, E. coli strains BL21(D3)> JM109> DH5α >M15, Archaebacteria, nematodes, Acinetobacter sp., Insect cells

S. cerevisiae, Schizo-saccharomyces pombe, C. albicans, B. subtilis, E. coli, P. pastoris, Strep. avidinii, Acinetobacter sp.

Yeast (S. cerevisiae), Acinetobacter sp.

Plant tissues (leaf, stem, root, seed) from many differentspecies (Arabidopsis, tobacco, maize, soybean, tomato,spinach, cotton, rice, fir, oak)

Sf9 and Sf21 insect cells (both adherent and in-suspension)

Cultured mammalian cells, COS-7, NIH 3T3, Hepa 1-6, 293, CHO, MDA, MB231, FM2 and Insect cells

Heart, liver, kidney, brain, stomach, lung, leg and pancreas

Tissue: calf liver.Cultured cells: epithelial (HeLa), fibroid (COS-7), kidney (NIH 3T3), liver (Hepa 1), brain (C6)

Cultured cells: brain (C6), epithelial (HeLa), fibroblasts (NIH 3T3), yeast (S. cerevisiae)

Mammalian cells

Liver, heart, kidney, and brain

Mammalian cells (Lysosome Kit only) or soft and hard tissues (all Kits)

Protein Extraction – Cell Lysis Selection GuideThermo Scientific Pierce Cell Lysis Reagents

1. Immunoprecipitation.2. Samples prepared in Mem-PER Reagent can be dialyzed if the buffer contains detergent (e.g., CHAPS).3. Thermo Scientific 2D Sample Prep for Nuclear Proteins (Product # PI-89863) and 2D Sample Prep for Membrane Proteins (Product # PI-89864) are designed using our popular

NE-PER and Mem-PER Reagents.

Protein Purification Technical Handbook This updated 79-page handbook provides techni-cal and product information to help maximizeresults for affinity purification procedures. Thehandbook provides background, helpful hints andtroubleshooting advice for covalent coupling ofaffinity ligands to chromatography supports,avidin:biotin-binding, affinity purification of anti-bodies, immunoprecipitation and co-immunopre-cipitation assays, affinity procedures for contami-nant removal, and related procedures.

Ordering Information

Product # Description Pkg. Size

Protease Inhibitor CocktailsPI-78425 Halt Protease Inhibitor Single-Use 24 x 100 μl

Cocktail, EDTA-free (100X)Each 100 µl microtube contains sufficientcocktail to treat 10 ml of lysate. Proteaseinhibitors prepared in DMSO solution.

PI-78430 Halt Protease Inhibitor Single-Use 24 x 100 μlCocktail (100X)Each 100 µl microtube contains sufficientcocktail to treat 10 ml of lysate. Proteaseinhibitors prepared in DMSO solution.

PI-78429 Halt Protease Inhibitor Cocktail 5 mlPI-78437 Halt Protease Inhibitor Cocktail, EDTA-free 5 mlPI-78438 Halt Protease Inhibitor Cocktail 10 mlPI-78439 Halt Protease Inhibitor Cocktail, EDTA-free 10 ml

Phosphatase Inhibitor CocktailsPI-78420 Halt Phosphatase Inhibitor Cocktail 1 ml

A mixture of four phosphatase inhibitors includingsodium fluoride, sodium orthovanadate, sodium pyrophosphate and β-glycerophosphate.

PI-78426 Halt Phosphatase Inhibitor Cocktail 5 x 1 mlPI-78427 Halt Phosphatase Inhibitor Cocktail 10 mlPI-78428 Halt Phosphatase Inhibitor Single-Use 24 x 100 μl

CocktailEach 100 µl microtube contains sufficient cocktail to treat 10 ml of lysate.

Combined Protease and Phosphatase Inhibitor CocktailsPI-78440 Halt Protease and Phosphatase 1 ml

Inhibitor CocktailPI-78441 Halt Protease and Phosphatase 1 ml

Inhibitor Cocktail, EDTA-freePI-78442 Halt Protease and Phosphatase 24 x 100 μl

Single-Use Inhibitor CocktailEach 100 µl microtube contains sufficient cocktail to treat 10 ml of lysate.

PI-78443 Halt Protease and Phosphatase 24 x 100 μl Inhibitor Single-Use Cocktail, EDTA-freeEach 100 µl microtube contains sufficient cocktail to treat 10 ml of lysate.

PI-78444 Halt Protease and Phosphatase 5 x 1 mlInhibitor Cocktail

PI-78445 Halt Protease and Phosphatase 5 x 1 mlInhibitor Cocktail, EDTA-free

PI-78446 Halt Protease and Phosphatase 10 mlInhibitor Cocktail

PI-78447 Halt Protease and Phosphatase 10 mlInhibitor Cocktail, EDTA-free

Individual InhibitorsPI-78431 AEBSF 100 mg

[4-(2-aminoethyl) benzenesulfonyl fluoride•HCl]

PI-78432 Aprotinin 25 mgPI-78433 Bestatin 10 mg

N-[(2S,3R)-3-amino-2-hydroxy-4-phenylbutyryl]-L-leucine

PI-78434 E-64 10 mg[N-trans-epoxysuccinyl-L-leucine-4-guanidinobuty-lamide]Also known as: N-[N-(L-3-trans-carboxirane-2-carbonyl)]-L-leucyl agmatin

PI-78435 Leupeptin 50 mg[acetyl-leucyl-leucyl-argininal]•hemisulfate

PI-78436 Pepstatin A 25 mgPI-36978 PMSF 5 g

phenylmethylsulfonyl fluoride

Cell Lysis Technical Handbook This 45-page handbook provides protocols, tech-nical tips and product information to help maxi-mize results for protein/gene expression studies.The handbook provides background, helpful hintsand troubleshooting advice for cell lysis, proteinpurification, cell fractionation, protease inhibitorsand protein refolding.

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch offi ce or distributor.

Thermo Scientifi c Pierce® Cell Lysis Technical HandbookFeaturing Cell Lysis Reagents and Detergents

The Halt Protease, Phosphatase and Combined Protease and Phosphatase Inhibitor Cocktails are compatible with all of theseThermo Scientific Pierce Cell Lysis Reagents.

© 2009 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are property ofThermo Fisher Scientific Inc. and its subsidiaries. B-PER Technology is protected by USPatent 6,174,704. Mitochondria Isolation Kit Technology is protected by US Patent 7,407,800.Slide-A-Lyzer MINI Dialysis Unit Technology is protected by US Patent 6,039,871. ThermoScientific Pierce Products are sold for research and manufacturing purposes only. Visit ourwebsite for up-to-date prices.

For customer service, call 1-800-234-7437To fax an order, use 1-800-463-2996To order online: www.fishersci.ca

To view a PDF or request a free copy of either handbook, log on to www.thermo.com/pierce or call 800-874-3723 or 815-968-0747. Outside the UnitedStates, contact your local branch office or distributor.

Our broad-spectrum protease and phosphatase inhibitor cocktails and individual protease inhibitors accommodate specific and general needs in cell lysis and protein extrac-tion methods.

• Thermo Scientific Halt Protease Inhibitor Cocktails target serine, cysteine and aspartic acid proteases andaminopeptidases. Metalloproteases are inhibited by theoptional addition of EDTA. Individual protease inhibitorstargeting separate classes of proteases are also avail-able for custom cocktail development.

• Halt™ Phosphatase Inhibitor Cocktails contain chemicalcompounds that target serine/threonine and tyrosinephosphatases.

• The Halt Protease and Phosphatase Inhibitor Cocktail prevents protein degradation and preserves phosphory-lation simultaneously, providing protection that is similar to the individual cocktails.

These inhibitors protect proteins during their extractionfrom cultured cells, animal tissues, plant tissues, yeast or bacteria. They are provided in a liquid format for easydispensing and are compatible with Thermo ScientificPierce Cell Lysis Reagents.

Product line highlights:

• Convenient storage – store refrigerated, saving valuable freezerspace

• Compatible with Pierce Cell Lysis Reagents

• Available in single-dose 1 ml, 5 ml, 5 x 1 ml and 10 ml packaging

Convenient and innovative single-use packagingAll of our protease and phosphatase inhibitor cocktails are availablein convenient single-use packaging. The single-use cocktails contain 24 x 100 μl foil-sealed microtubes sufficient to treat 240 ml of cellextract. As a 100X concentrate, each single-use tube provides the volume needed to inhibit protease or phosphatase activity up to 10 mlof cell lysate.

The foil seal of each microtube is easily punctured by a pipette tip.Only the volume that yields a 1X concentration of the componentinhibitors is typically needed. The remaining tubes are preserved from exposure to air, moisture and the potential for contamination.

Single-use, ready-to-use packaging: • Keeps inhibitors preserved between uses

• Reduces potential of cocktail contamination

• Minimizes waste by using only the amount of cocktail needed

• Allows immediate lysate inhibition with no thawing or waiting fortablets to dissolve

Applications for protease and phosphatase inhibitorcocktails:

• Protecting lysates produced by a variety of methods, includingPierce Cell Lysis Reagents, sonication, French press, blenderhomogenization or repetitive freeze/thaw cycling

• Purifying proteins via immobilized metal chelate affinity chromatog-raphy (IMAC) or other downstream procedures where EDTA wouldinterfere

• Screening extracts for proteolytic activity

• Studying proteolysis in the regulation of cellular processes

• Preserving phosphorylation states of samples in biological pathwaystudies

Halt Protease Inhibitor Cocktails

Stops proteolysis fast with a cocktail that is delivered toyour sample within seconds

Waiting for an inhibitor tablet to dissolve leaves your lysate unprotectedand at risk for protein degradation. This vulnerability is especially criticalwhen the protein target is in low abundance. The single-use solution for-mat allows you to deliver the appropriate amount of inhibitor for the vol-ume of lysate in an instant.

With the Halt Inhibitor Cocktails there is no waiting for a tablet to dis-solve, no sample vortexing required, no imprecise tablet splitting and,most importantly, no potential loss of valuable target while waiting for the tablet to dissolve.

The Halt Inhibitor Cocktail is simply withdrawn from the tube and addeddirectly to the lysate sample within seconds. Comparatively, it takesapproximately two minutes to fully dissolve a tablet-based cocktail,exposing your valuable protein to proteolytic attack. When tested ona pancreatic tissue lysate, the ready-to-use formulation significantlyout-performed the tablet in proteolytic activity knockdown and sampledelivery speed (Figure 1).

Protease inhibitor highlights:• Effective for suppressing proteolytic activity in detergent-based cell

lysis reagents

• Same high-quality protease inhibitor components available separately

• Prepare a custom cocktail or increase the concentration of a specificprotease inhibitor in the formulation

• EDTA-free formulation inhibits proteolytic activity in applications inwhich EDTA may interfere with protein stability, subsequent assays orpurification methods such as immobilized metal chelate affinity chro-matography (IMAC)

Halt Protease Inhibitor Cocktails are provided in ready-to-use formula-tions that inhibit serine, cysteine and aspartic acid proteases and metalloproteases. We have demonstrated outstanding performance withThermo Scientific M-PER Mammalian, B-PER Bacterial, T-PER Tissue andY-PER Yeast Protein Extraction Reagents (Figure 2). Add the appropriatevolume of inhibitor cocktail to the sample (10 μl per 1 ml of cell extract)and proceed directly to protein purification. The EDTA-free formulation isideal for preparing samples that will be analyzed by 2D gel electrophore-sis. As a 100X concentrate in DMSO, the cocktail contains reversible andirreversible inhibitors of serine, cysteine, metalloproteases, and asparticacid proteases in addition to aminopeptidases present in virtually all celllysate samples.

Control;Extract Only

Protease InhibitorCocktail Tablet

Thermo Scientific HaltSingle-Use Cocktail

Tablet;EDTA-Free

Thermo Scientific HaltSingle-Use EDTA-Free

Cocktail

Percent Inhibition

Figure 1. Thermo Scientific Halt Protease Inhibitor Single-use Cocktails are more effective than tablet-format cocktails. Using a validated protease assay and 1.0 mg/ml of rat pancreas extract, the Halt Protease Inhibitor Single-UseCocktails, with and without EDTA added, were tested against commercially available tablet-format protease inhibitor cocktails under the same conditions. A 1X final concentration of each inhibitor was added. The single-use formulationresulted in ≥ 97% inhibition compared to ≥ 59% inhibition for the tablet.

Perform protein extractions and purifications with confidence

1 2 3 4 1 2 3 4

A. Thermo Scientific B-PERBacterial Protein Extraction Reagent

B. Thermo Scientific M-PERMammalian Protein Extraction Reagent

Figure 2. Thermo Scientific Halt Protease Inhibitor Single-Use Cocktails are compatible with Thermo Scientific M-PER Mammalian Protein Extraction and B-PER Bacterial Protein Extraction Reagents. Bovine serum albumin (BSA) was incubated overnight at 37°C in the presence of 0.1 mg/ml trypsin in M-PERExtraction Reagent and B-PER Extraction Reagent, respectively. Under extremetime and temperature conditions, substantial protection from degradation wasobserved as marginal to barely detectable cleavage products of BSA. Lane 1. Control 1, BSA only; Lane 2. Control 2, Trypsin (0.1 mg/ml) (+) BSA and no Halt Protease Inhibitor Single-Use Cocktail present; Lane 3. BSA (+) Trypsinwith Halt Protease Inhibitor Single-Use Cocktail with EDTA; and Lane 4. BSA (+)Trypsin with Halt Protease Inhibitor Single-Use Cocktail.

Identity of each component of the Halt Protease Inhibitor Cocktail is readily availableTable 1. Formulation and concentrations of the Thermo Scientific Halt Protease Inhibitor Cocktails.

Concentration of Each Inhibitor in theProtease Protease Inhibitor Solubility Protease Inhibitor Cocktails (100X in DMSO) Inhibitor MW Family Targeted Type (Solvent System) (Product # PI-78430 and PI-78425)AEBSF•HCl 239.5 Serine proteases Irreversible 200 mg/ml (H2O) 100 mMAprotinin 6511.5 Serine proteases Reversible 10 mg/ml (H2O) 80 μMBestatin 308.38 Amino peptidases Reversible 5 mg/ml (methanol) 5 mME-64 357.4 Cysteine proteases Irreversible 20 mg/ml (1:1 EtOH/H2O) 1.5 mMEDTA (not included 372.24 Metalloproteases Reversible 10 g/100 ml (H2O) 0.5 Min Product # PI-78425) (Chelates divalent cations)Leupeptin 475.6 Serine and cysteine proteases Reversible 1 mg/ml (H2O) 2 mMPepstatin A 685.9 Aspartic acid proteases Reversible 1 mg/ml (MeOH) 1 mM

Halt Combined Protease and PhosphataseInhibitor Cocktails

Provides complete protection from proteases and phosphatases in one reliable cocktail

The all-in-one Halt Protease and Phosphatase Inhibitor Cocktail providessingle-solution convenience with full sample protection. This broad-spec-trum inhibitor cocktail is specifically optimized to protect proteins fromdegradation during extraction and purification.

The cocktail contains inhibitors against the major classes of proteasesand phosphatases, targeting aminopeptidases, cysteine and serine pro-teases, and serine/threonine and tyrosine phosphatases (Table 2).

This combined cocktail is the first of its kind. The aqueous-based formatallows for convenient cold-room storage without freezing and is mucheasier to use than tablets that require tedious splitting with a razor bladeto treat samples < 10 ml. More importantly, the cocktail formulation ismore effective than the tablet format (Figure 4) at inhibiting proteases inlysates and preserving phosphorylation during cell harvest. The formula-tion is also mass spectrometry-compatible because it does not containAEBSF, which can cause peaks to shift.

Combined cocktail highlights:• Convenient all-in-one cocktail contains both protease and phos-

phatase inhibitors

• Compatible with Pierce Cell Lysis Reagents and mass spectrometry

• Identity of each component of the cocktail is listed

Table 2. Inhibitors included in the Thermo Scientific Halt Protease andPhosphatase Inhibitor Cocktail formulation and their targeted enzyme class.

Inhibitor TargetSodium Fluoride Ser/Thr and Acidic PhosphatasesSodium Orthovanadate Tyr and Alkaline Phosphatasesβ-glycerophosphate Ser/Thr PhosphatasesSodium Pyrophosphate Ser/Thr PhosphatasesAprotinin Ser ProteasesBestatin Amino-peptidasesE64 Cysteine ProteasesLeupeptin Ser/Cys ProteasesEDTA (optional) Metalloproteases

% Inhibition

Control; No InhibitorSupplier R Tablet:Protease InhibitorCocktail EDTA-Free

Halt Protease &PhosphataseInhibitor CocktailEDTA-FreeSupplier R Tablet:Protease InhibitorCocktail + EDTA

Halt Protease &PhosphataseInhibitor Cocktail +EDTA

Halt Phosphatase Inhibitor Cocktails

Safeguards against serine, threonine and tyrosine phosphatase activitiesPhosphorylation and dephosphorylation is a molecular on/off switch thatregulates a number of key biological pathways within the cell, includingsignal transduction, cell division and apoptosis. The Halt PhosphataseInhibitor Cocktail preserves protein phosphorylation in cell lysates andtissue extracts. The cocktail contains a mixture of four phosphataseinhibitors of broad specificity, including sodium fluoride, sodium ortho-vanadate, sodium pyrophosphate and β-glycerophosphate. Unlike other commercially available phosphatase inhibitor cocktails that protect against either serine/threonine phosphatases or protein tyrosinephosphatases, the Halt Phosphatase Inhibitor Cocktail protects phospho-proteins from both families of phosphatases (Figure 3).

Phosphatase inhibitor highlights:• Complete protection from phosphatases – inhibits both serine/threo-

nine and protein tyrosine phosphatases

• Compatible with standard protein assays – quantify treated samples with the Thermo Scientific Pierce BCA Protein Assay Kit, Coomassie Plus (Bradford) Assay or Pierce 660 nm Protein Assay

• 1 ml protects up to 100 ml of sample

A. MEK+– +–

B. MAPK+– +–

C. STAT3+– +–

PSTAT3

Figure 3. Halt Phosphatase Inhibitor Cocktail preserves phosphorylation of MEK,MAP Kinase 42/44 and STAT3 in HeLa cell lysate. HeLa cells were lysed in theabsence (-) and presence (+) of a 1X concentration of the Halt PhosphataseInhibitor cocktail. Cell lysates were analyzed by Western blot for total and phos-phorylated protein as indicated. Panel A. MEK and phosphorylated MEK (PMEK), Panel B. MAP Kinase and PMAP Kinase and Panel C. STAT3 and PSTAT3. The proteins are phosphorylated on serine, threonine/tyrosine and tyrosine, respectively.

Figure 4. Thermo Scientific Halt Protease and Phosphatase Inhibitor Cocktails aremore effective than tablet-format protease inhibitor cocktails. Using a general protease assay with a rat pancreas extract (1 mg/ml), the Halt Protease and Phos-phatase Inhibitor Cocktails (± EDTA) were compared with the tablet-based proteaseinhibitor cocktails (± EDTA) of Supplier R’s Tablet. The same experimental conditionswere used for all samples tested. A 1X final concentration of each inhibitor cocktailwas added to the extract.

lysis reagents

Tablet;EDTA-Free

Cocktail

Percent Inhibition

1 2 3 4 1 2 3 4

phatase inhibitors

% Inhibition

A. MEK+– +–

B. MAPK+– +–

C. STAT3+– +–

PSTAT3

lysis reagents

Tablet;EDTA-Free

Cocktail

Percent Inhibition

1 2 3 4 1 2 3 4

phatase inhibitors

% Inhibition

A. MEK+– +–

B. MAPK+– +–

C. STAT3+– +–

PSTAT3

Product

P-PER® KitPI-89803

For TissuePI-89801

Compatibility

Organisms/Samples

Mammalian cells

Product

P-PER® KitPI-89803

For TissuePI-89801

Compatibility

Organisms/Samples

Mammalian cells

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