Protease Inhibitor Cocktail (ab65621)

Abcam's Protease Inhibitor Cocktail contains a mixture of water-soluble protease inhibitors with broad specificity for various proteases, and has been optimized to generate the maximal protection to mammalian tissue and cell extracts.

Supplied as a lyophilized powder.

One vial can make 125 ml of cocktail solution.

Add 250 µl DMSO to the vial, pipette several times to dissolve all powder (This makes 500X concentrated inhibitor cocktail).

Visit our FAQs page for tips and troubleshooting.

Crude cell extracts contain many endogeneous enzymes, such as proteases and phosphotases, which can degrade valuable proteins in the extract. The individual components of the protease inhibitor cocktail have specific inhibitory properties. A description of each inhibitor is given below:

Aprotinin: Inhibits serine proteases, such as trypsin, chymotrypsin, plasmin, trypsinogen, urokinase, and kallikrein.

Leupeptin: Inhibits both serine and cysteine proteases, such as calpain, trypsin, papain, and cathepsin B.

Pepstain A: Inhibits acid proteases, such as pepsin, rennin, cathepsin D, chymosin, and protease B.

PMSF: Inhibits serine proteases, such as trypsin and chymotrypsin. Also inhibits cysteine proteases and mammalian acetylcholinesterase.

Properties

Aprotinin
Leupeptin
Pepstatin
PMSF

References

This product has been referenced in:

Wang B et al. Essential control of mitochondrial morphology and function by chaperone-mediated autophagy through degradation of PARK7. Autophagy12:1215-28 (2016). Read more (PubMed: 27171370) »
Knight WE et al. PDE1C deficiency antagonizes pathological cardiac remodeling and dysfunction. Proc Natl Acad Sci U S AN/A:N/A (2016). Read more (PubMed: 27791092) »

Publishing research using ab65621? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Protease and Phosphatase Inhibition

Protease and phosphatase inhibitor cocktails and tablets are ideal for the protection of proteins during extraction or lysate preparation from primary cells, cultured mammalian cells, animal tissues, plant tissues, yeast or bacterial cells. Individual protease inhibitors are also available separately and in multiple sizes. Formulations with or without EDTA are available for divalent cation-sensitive assays.

Effective—outperforms leading competitor formulations across targeted protease and phosphatase families
Multiple package sizes—liquid cocktails and tablets are available in multiple sizes and formats to accommodate different volume and pricing needs
Improved formulation—tablets dissolve more quickly into a clear solution, and are fully compatible with all Pierce protein assays.
Convenient—refrigerator-stable formulations are easier to use than individual inhibitors
Complete protection—all-in-one formulations contain both protease and phosphatase inhibitors (combined cocktail only)
Compatible—does not interfere with protein assays or other downstream applications

Protease Inhibitor Cocktail

for use with mammalian cell and tissue extracts, DMSO solution

Popular Documents: Datasheet (PDF) | Specification Sheet (PDF)

Properties

Description

Frequently Asked Questions

Frequently Asked Questions are available for this Product.

Components

Biochem/physiol Actions

This mixture contains individual components, including AEBSF at 104 mM, Aprotinin at 80 μM, Bestatin at 4 mM, E-64 at 1.4 mM, Leupeptin at 2 mM and Pepstatin A at 1.5 mM. Each component has specific inhibitory properties. AEBSF and Aprotinin act to inhibit serine proteases, including trypsin, chymotrypsin, and plasmin amongst others. Bestatin inhibits aminpeptidases. E-64 acts against cystein proteases. Leupeptin acts against both serine and cystein proteases. Pepstatin A inhibits acid proteases.

This cocktail should be stored at -20 °C.

1, 5 mL in serum bottle

Preparation Note

One mL of this cocktail solution is recommended for the inhibition of endogenous enzymes found in 100 mL of lysate from 20 g of bovine liver or 10 mL of lysate from CHO cells. This product is supplied as a solution in DMSO.

Specificity

Inhibits serine, cysteine, and acid proteases, and aminopeptidases.

Application

Protease Inhibitor Cocktail has been used-

• as a buffer component during sonication of GFP (green fluorescent protein)-huntingtin-trans fected HEK 293 cells in GST (glutathione S-transferase) pull down assay

• as a component of lysis buffer

• as a component of radioimmunoprecipitation assay buffer (RIPA)

Price and Availability

Safety Information

Certificate of Analysis

Certificate of Origin

Frequently Asked Questions

The Sigma-Aldrich portfolio of protease inhibitor cocktails shows an excellent track record in the peer-reviewed scientific literature, as witnessed by the many publications that cite our protease in.

Keywords: AGE, Adhesion, Aerobic, Affinity chromatography, Alzheimer Disease, Amplification, Angiogenesis, Anti-inflammatory agents, Antibiotics, Antimicrobials, Antitumor agents, Antivirals, Apoptosis, Atomic absorption spectroscopy, Autophagy, Biochemistry, Biological processes, Buffers, Cancer, Cardiovascular, Catalysis, Cell biology, Cell disruption, Cell proliferation, Cell signaling, Cellular processes, Central Nervous System, Chromatin immunoprecipitation, Chromatography, Clinical, Cloning, Cytotoxins, DNA replication, Degradations, Detergents, Diabetes, Diagnostic, Diseases, Dopamine agents, Drug discovery, Eliminations, Environmental, Enzyme-link immunosorbent assay, Gas chromatography, Gastrointestinal, Gene expression, Genetic, Genetics, Gluconeogenesis, Glycosylations, Growth factors, Hormones, Hydration reaction, Hydroxylations, Immobilization, Immunology, Immunoprecipitation, Inflammation, Ligands, Lipid Metabolism, Lipid peroxidation, Mass spectrometry, Medicinal chemistry, Metabolism, Metabolites, Methylations, Microarray Analysis, Microbiology, Microscopy, Molecular biology, Morphogenesis, Neurology, Neuroscience, Neurotransmission, Neurotransmitters, Normal-phase chromatography, Nutrition, Obesity, Oncology, PAGE, Parkinson Disease, Peroxidations, Pharmaceutical, Phosphorylations, Photosynthesis, Physiological Processes, Polymerase chain reaction, Polymorphisms, Proteomics, Purification, Recombination, Reductions, Reproduction, Respiratory, Reversible addition-fragmentation chain transfer polymerizations, Sample preparations, Scanning electron microscopy, Schizophrenia, Separation, Sequencing, Sonication, Transcription, Transduction, Veterinary, Vitamins, Western blot, transformation

References

also commonly purchased with this product

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Protease Inhibitor Cocktail (100X) #5871

Gallery: Protease Inhibitor Cocktail (100X) #5871

Product Description

When diluted in lysis buffer to a final concentration of 1X the Protease Inhibitor Cocktail prevents protein degradation by endogenous proteases present in whole cell extract. The 100X

Protease Inhibitor Cocktail is a clear, colorless liquid.

Product Usage Information

1. Briefly vortex the Protease Inhibitor Cocktail (100X) before use.

2. Just prior to lysing cells, dilute the cocktail 1:100 in desired lysis buffer to obtain a 1X working concentration.

Solutions and Reagents:

The Protease Inhibitor Cocktail (100X) is composed of a proprietary mix of AEBSF, Aprotinin, Bestatin, E64, Leupeptin, and Pepstatin A to promote broad spectrum protection against endogenous proteases. The cocktail does not contain EDTA (a metalloprotease inhibitor) which can be incompatible with some downstream applications (i.e. protein assays, 2D electrophoresis, etc.). If EDTA is desired as a protease inhibitor it can be added to the cell lysis buffer at a final working concentration of 5 mM.

Storage: Store the undiluted 100X cocktail at 4ºC. Do not freeze.

Background

In order to study specific target proteins of interest protease-mediated degradation during the generation of protein lysates is to be avoided. A loss of normal cellular control occurs during cell lysis, and endogenous proteases within the cell extract are free to degrade proteins in an uncontrolled manner. The addition of protease inhibitors to the cell lysis buffer aids in the preservation of target proteins in the cell extract.

Data Sheets & Documentation

For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

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Protocol: Protease and Phosphatase Inhibitors

Protease Inhibitors:

Protease Inhibitor Cocktails:

These are available from many companies. The choice of cocktail depends on the species of sample with which you are working. The cocktails also vary on the presence or absence of EDTA. EDTA is a valuable protease inhibitor, but should be avoided if benzonase is used in sample preparation or if phosphopeptides are to be analyzed. The cocktail may be in a concentrated solution, usually dissolved in DMSO, or a tablet.

Thermo Scientific

Halt Protease Inhibitor Cocktail - AEBSF, aprotinin, bestatin, E-64, leupeptin and pepstatin A, with EDTA (catalog no. 78430) or no EDTA (catalog no. 78425)

Sigma carries a wide variety of cocktails for various species; a useful one is catalog no. P8340

GE Healthcare

Protease inhibitor cocktail, catalog no. GE80-6501-23, has no EDTA

Complete protease Inhibitor cocktail tablets, EDTA free, 1 tablet for 10 ml, catalog no. 11836170001

Protease Inhibitor Cocktail, a powder that can be dissolved in DMSO to make a 500 x solution. Contains Aprotinin, Leupeptin, Pepstatin, PMSF. Catalog no. ab65621

Phosphatase Inhibitors:

Phosphatase Inhibitor Cocktails:

Phosphatase Inhibitor Cocktail 2 - sodium vanadate, sodium molybdate, sodium tartrate, and imidazole (catalog no. P5726)
Phosphatase Inhibitor Cocktail 3- cantharidin, p-bromolevamisole oxalate, and calyculin A (catalog no. P0044)

Sigma recommends using Cocktail 2 and Cocktail 3 in combination

EMD Millipore

Phosphatase Inhibitor Cocktail Set I (catalog no. 524624) - (-)-p-bromotetramisole, cantharidin, microcystin LR
Phosphatase Inhibitor Cocktail Set II (catalog no. 524625) - imidazole, NaF, sodium molybdate, sodium orthovanadate, sodium tartrate

Thermo Scientific

Halt Phosphatase Inhibitor Cocktail - sodium fluoride, sodium orthovanadate*, sodium pyrophosphate and beta-glycerophosphate. Thermo Scientific 78428
Halt Protease and Phosphatase Inhibitor Cocktail - sodium fluoride, sodium orthovanadate*, sodium pyrophosphate and beta-glycerophosphate, aprotinin, bestatin, E-64, leupeptin (Thermo catalog no. 78442, with EDTA or catalog no. 78443, without EDTA)

*Sodium orthovanadate preparation

All steps to be performed in a fume hood.

Prepare a 100 mM solution in double distilled water.
Set pH to 9.0 with HCl.
Boil until colorless. Minimize volume change due to evaporation by covering loosely.
Cool to room temperature.
Set pH to 9.0 again.
Boil again until colorless.
Repeat this cycle until the solution remains at pH 9.0 after boiling and cooling.
Bring up to the initial volume with water.
Store in aliquots at - 20°C. Discard if samples turn yellow

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Protease Inhibitor Cocktails

Don't Lose Your Proteins During Sample Prep

Optimized to maintain and preserve protein functionality following cell lysis
Available in powder, solution, or tablet form
Inhibits proteases in almost any tissue or cell
Specific formulations optimized for particular applications

Our Inhibitor Cocktails have a stellar performance record, as seen in reference citations from our customers.

View additional citations for each individual protease inhibitor cocktail product under the "Peer-Reviewed Papers" tab of each product’s detail page.

Specificity of Inhibition

Aprotinin, 0.3 μM

Bestatin, 130 μM

Pepstatin A, 2.2 mM

1,10-Phenanthroline, 500 mM

Phosphatase inhibitors: tyrosine, serine/threonine, acid and alkaline phosphatases

• Phosphoramidon disodium salt

Halt™ Protease Inhibitor Cocktail (100X)

Каталожный номер: 78430

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Часто задаваемые вопросы

Цитаты и ссылки

• No proprietary ingredients—fully disclosed formulation contains optimized concentrations of six broad-spectrum protease inhibitors AEBSF, aprotinin, bestatin, E-64, leupeptin and pepstatin A stabilized in high-quality dimethylsulfoxide (DMSO)

• Multiple package sizes—choose among several package sizes, including single-use microtubes that keep protease inhibitors fresh and reduce the risk of reagent contamination

• Regular formulation—includes a separate vial of EDTA solution for optional metalloprotease inhibition

• Lysis buffer compatible—use with Thermo Scientific Pierce Cell Lysis Buffers or nearly any other commercial or homemade detergent-based lysis reagent

• Protection of intact, active cellular proteins from degradation by endogenous proteases

• Inhibition of protease activity from lysates produced by a variety of methods including Thermo Scientific Pierce Cell Lysis Reagents, other commercially formulated cell or tissue lysis products, sonication, French press, blender homogenization or repetitive freeze/thaw cycling

• Screening of extracts for proteolytic activity

• The study of proteolysis in the regulation of cellular processes

Using a validated protease assay and 1.0mg/mL of rat pancreas extract, the Thermo Scientific Halt Protease Inhibitor Single-Use Cocktails were tested against other commercially available tablet-format protease inhibitor cocktails under the same conditions. A 1X final concentration of each inhibitor was added. The single-use formulation resulted in ≥97% inhibition compared to ≥59% inhibition for the tablet." data-omni-action="View figure">

Performance comparison of commercial protease inhibitor cocktails and tablets

Formulation and concentration of the Thermo Scientific Halt Protease Inhibitor Cocktail

Спецификации

Содержимое и хранение

Руководства и протоколы

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Protease Inhibitor Cocktail I

Biological Activity

Blend of protease inhibitors optimized for mammalian cells. Contains the following inhibitors: AEBSF HCl (100 mM), Aprotinin (80 μ M), Bestatin (5 mM), E-64 (1.5 mM), Leupeptin (2 mM) and Pepstatin (1 mM). Cocktail is provided as 1 ml of 100X concentrate in DMSO.

Technical Data

The technical data provided above is for guidance only. For batch specific data refer to the Certificate of Analysis.

All Tocris products are intended for laboratory research use only.

Solubility Data

Product Datasheets

References

References are publications that support the products' biological activity.

If you know of a relevant reference for Protease Inhibitor Cocktail I, please let us know.

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Keywords: Protease Inhibitor Cocktail I, supplier, AEBSF, Aprotinin, E-64, Bestatin, Leupeptin, Pepstatin, Biochemicals, and, Molecular, Biology, Biochemicals, and, Molecular, Biology, Tocris Bioscience

1 Citation for Protease Inhibitor Cocktail I

Citations are publications that use Tocris products. Selected citations for Protease Inhibitor Cocktail I include:

Fitting et al (2014) Interactive HIV-1 Tat and morphine-induced synaptodendritic injury is triggered through focal disruptions in N+ influx, mitochondrial instability, and Ca2+ overload. J Biol Chem 34 12850 PMID: 25232120

Do you know of a great paper that uses Protease Inhibitor Cocktail I from Tocris? If so please let us know.

Protease Inhibitor Cocktail I Reviews

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Protease/Phosphatase Inhibitor Cocktail (100X) #5872

Western blot analysis of extracts from NIH/3T3 cells, prepared in lysis buffer in the absence of protease inhibitors (left) or with Protease/Phosphatase Inhibitor Cocktail (100X) #5872 added (right), and incubated at 37ºC for the indicated time points, using β-Catenin (D10A8) XP® Rabbit mAb #8480. In the absence of protease inhibitors, β-Catenin signal fades within 3 hr after harvest, indicating protein degradation. In the presence of the protease inhibitor cocktail, the β-Catenin degradation is slowed significantly and signal is still present at 20 hr following harvest.

Western blot analysis of extracts from NIH/3T3 cells, serum-starved overnight and treated with hPDGF-BB #8912 (100ng/ml, 5min), prepared in lysis buffer in the absence of phosphatase inhibitors (left) or with Protease/Phosphatase Inhibitor Cocktail (100X) #5872 added (right), and incubated at 37ºC for the indicated time points, using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower). In the absence of phophatase inhibitors, phospho-Akt signal drops significantly after time point 0, demonstrating rapid loss of phosphorylation at later time points. In the presence of the phosphatase inhibitor cocktail, the phospho-Akt signal is preserved through all time points monitored.

Gallery: Protease/Phosphatase Inhibitor Cocktail (100X) #5872

Product Description

When diluted in lysis buffer to a final concentration of 1X the Protease/Phosphatase Inhibitor Cocktail prevents protein degradation and dephosphorylation by endogenous proteases and phosphatases present in the whole cell extract. The 100X cocktail is a clear light yellow to light green liquid.

Product Usage Information

1. Briefly vortex the Protease/Phosphatase Inhibitor Cocktail (100X) before use.

2. Just prior to lysing cells, dilute the cocktail 1:100 in desired lysis buffer to obtain a 1X working concentration.

Solutions and Reagents:

The Protease/Phosphatase Inhibitor Cocktail (100X) is composed of a proprietary mix of Aprotinin, Bestatin, E64, and Leupeptin to promote broad spectrum protection against endogenous proteases and sodium fluoride, sodium pyrophosphate, β-glycerophosphate, and sodium orthovanadate to promote broad spectrum protection against endogenous serine/threonine and tyrosine phosphatases. The cocktail does not contain EDTA (a metalloprotease inhibitor) which can be incompatible with some downstream applications (i.e. protein assays, 2D electrophoresis, etc.). If EDTA is desired as a protease inhibitor it can be added to the cell lysis buffer at a final working concentration of 5mM.

Storage: Store the undiluted 100X cocktail at 4ºC. Do not freeze. This product is stable for 12 months.

Background

Dynamic protein phosphorylation is a key cellular signaling mechanism by which a broad spectrum of cellular processes is regulated. In order to study the phosphorylation status of specific target proteins the phosphorylated residue of interest must remain intact. When cells are lysed to make whole cell extracts, a loss of normal cellular signaling regulation occurs, and phosphatases within the cell extract are free to dephosphorylate proteins in an uncontrolled manner. The addition of phosphatase inhibitors to the cell lysis buffer aids in the preservation of phosphorylated residues at the time of cell disruption.

This same loss of normal cellular control when generating whole cell extracts also leads to uncontrolled degradation of proteins by endogenous proteases. The addition of protease inhibitors to the cell lysis buffer aids in the preservation of target proteins in the cell extract.

Data Sheets & Documentation

For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

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воскресенье, 4 февраля 2018 г.