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539158 20S Proteasome Assay Kit, SDS-Activated

20S Proteasome Assay Kit, SDS-Activated MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
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Áttekintés

Replacement Information

Kulcsspecifikációk táblázata

Detection Methods
Fluorometric
Description
Overview

This product has been discontinued.



Useful in quantifying 20S proteasome activity in cuvettes or a ½ volume 96-well plate. 20S activity is measured by monitoring the release of free AMC from the fluorogenic peptide Suc-Leu-Leu-Val-Tyr-AMC over time.
Catalogue Number539158
Brand Family Calbiochem®
Materials Required but Not Delivered• Microcentrifuge
• Fluorescence spectrophotometer
• Quartz cuvettes or flat-bottom opaque 96-well plate
• Pipetman (0-20 µl; 100 µl and 500 µl); multi-channel pipeteman (if using 96-well configuration)
• AMC (7-Amino-4-methylcoumarin), 10 mg (M.W. 175.2; Calbiochem Cat. No. 164545). Supplied as a lyophilized solid.
• DMSO (to dissolve the AMC)
References
ReferencesDeMartino, G.N., and Slaughter, C. 1999. J. Biol. Chem. 274, 22123.
Adams, G., et al. 1998. Biochemistry 37, 12927.
Glickman, M., et al. 1998. Cell 94, 615.
Groll, M., et al. 1997. Nature 386, 463.
Coux, O., et al. 1996. Annu. Rev. Biochem. 65, 801.
Stein, R. et al. 1996. Biochemistry 35, 3899.
Seemuller, E., et al. 1995. Science 268, 579.
Shibatani, T., and Ward, W. 1995. Arch. Biochem. Biophys. 321, 160.
Hoffman, L., et al. 1992. J. Biol. Chem. 267, 22362.
Ma, C.P., et al. 1992. J. Biol. Chem. 267, 10515.
Product Information
Detection methodFluorometric
FormatCuvette or 96-well plate
Kit contains20S Proteasome in Buffer, Reaction Buffer, SDS, Substrate Solution, and a user protocol. Does not include the 7-Amino-4-methylcoumarin (AMC; Cat. No. 164545) standard.
Quality LevelMQ100
Applications
Biological Information
Assay time30 min
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
R PhraseR: 36/37/38-22

Irritating to eyes, respiratory system and skin.
Harmful if swallowed.
S PhraseS: 26-36

In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
Wear suitable protective clothing.
Product Usage Statements
Storage and Shipping Information
Ship Code Dry Ice Only
Toxicity Multiple Toxicity Values, refer to MSDS
Storage Multiple storage requirements
Storage ConditionsUpon arrival of the kit store the Enzyme Solution -80°C. The remainder of the kit should be stored at -20°C.
Avoid freeze/thaw Avoid freeze/thaw
Do not freeze Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit contains20S Proteasome in Buffer, Reaction Buffer, SDS, Substrate Solution, and a user protocol. Does not include the 7-Amino-4-methylcoumarin (AMC; Cat. No. 164545) standard.
Specifications
Global Trade Item Number
Katalógusszám GTIN
539158 0

Documentation

20S Proteasome Assay Kit, SDS-Activated Certificates of Analysis

TitleLot Number
539158

References

Hivatkozások áttekintése
DeMartino, G.N., and Slaughter, C. 1999. J. Biol. Chem. 274, 22123.
Adams, G., et al. 1998. Biochemistry 37, 12927.
Glickman, M., et al. 1998. Cell 94, 615.
Groll, M., et al. 1997. Nature 386, 463.
Coux, O., et al. 1996. Annu. Rev. Biochem. 65, 801.
Stein, R. et al. 1996. Biochemistry 35, 3899.
Seemuller, E., et al. 1995. Science 268, 579.
Shibatani, T., and Ward, W. 1995. Arch. Biochem. Biophys. 321, 160.
Hoffman, L., et al. 1992. J. Biol. Chem. 267, 22362.
Ma, C.P., et al. 1992. J. Biol. Chem. 267, 10515.

Brochure

Title
Caspases and other Apoptosis Related Tools Brochure
Proteasome/Ubiquitination NF-kB Pathway Brochure
User Protocol

Revision10-June-2008 JSW
FormatCuvette or 96-well plate
Detection methodFluorometric
StorageUpon arrival of the kit store the Enzyme Solution -80°C. The remainder of the kit should be stored at -20°C.
BackgroundProteasomes are large multi-subunit complexes that selectively degrade intracellular proteins. Proteasomes are localized both in the nucleus and in the cytoplasm and associate with the membrane of the endoplasmic reticulum. Proteasome structure formation is characterized by the initial formation of an α-ring matrix, which provides the docking sites for a defined subset of β-subunits. Subsequently, the subunits bind and dimerization of two half-proteasomes occurs. A protein marked for degradation is attached to multiple molecules of ubiquitin, a 76-amino acid protein, which targets the protein for rapid hydrolysis by the 26S proteasome, The proteolytic core of this complex consists of the 20S proteasome and a 18S regulatory complex composed if multiple ATPases. The 20S proteasome contains multiple peptidase activities and has a molecular mass of about 700 kDa. In the ubiquitin-proteasome degradation pathway, ubiquitin is activated by an activating enzyme and is transferred to the conjugating enzyme. The conjugating enzyme helps attach ubiquitin to the e-amino groups of lysine residues of substrate proteins. In this process, a ubiquitin-ligating enzyme may also be involved. Ubiquitinated protein is escorted to the proteasome where it undergoes final degradation and the ubiquitin is recycled.
Principles of the assayThe Calbiochem® 20S Proteasome Assay Kit, SDS-Activated contains buffers and reagents for the quantitative analysis of 20S proteasome activity in cuvettes or 96-well plate. The 20S activity is measured by monitoring the release of free AMC from the fluorogenic peptide Suc-Leu-Leu-Val-Tyr-AMC. The rate of AMC release may be measured by monitoring the increase in fluorescence over time (excitation max.: ~380 nm; emission max.: ~460 nm).
Materials provided• 20X Reaction Buffer (Kit Component No. KP8601-1.5ML): 1 vial, 1.5 ml; supplied as 500 mM HEPES, 10 mM EDTA, pH 7.6
• 100X Activation Solution (Kit Component No. KP8602-125UL): 1 vial, 125 µl; supplied as 3% SDS (w/v) in H₂O
• 1000X Substrate Solution (Kit Component No. KP8603-25UL): 1 vial, 25 µl; supplied as 10 mM Suc-Leu-Leu-Val-Tyr-AMC in DMSO
• Enzyme Solution (Kit Component No.KP8604-12.5UG): 1 vial, 12.5 µg; supplied as 20S Proteasome at 1 mg/ml in 50 mM HEPES, pH 7.6, 50 mM NaCl, 1 mM DTT. NOTE: The 20S Proteasome solution as supplied is stable on ice for at least 24 h. Once activated with SDS, the activity is stable at 37°C for at least 1 h.
Materials Required but not provided• Microcentrifuge
• Fluorescence spectrophotometer
• Quartz cuvettes or flat-bottom opaque 96-well plate
• Pipetman (0-20 µl; 100 µl and 500 µl); multi-channel pipeteman (if using 96-well configuration)
• AMC (7-Amino-4-methylcoumarin), 10 mg (M.W. 175.2; Calbiochem Cat. No. 164545). Supplied as a lyophilized solid.
• DMSO (to dissolve the AMC)
Precautions and recommendations• For quantitative measurements of the proteasome, the fluorimeter may be calibrated by generating a standard curve using AMC. The standard curve should have a range of 0-100 pmoles AMC. This calibration allows for the calculation of exact specific activity of the 20S proteasome on each individual fluorimeter. For example, make a 10 µM stock solution of AMC in DMSO, prepare two-fold serial dilutions, starting with 10 µM down to 0 µM, and read the fluorescence.
• For optimum activity, it is recommended that the assay be performed at 37°C. However, if the measurement instrument has no temperature control, the assay may be performed at room temperature. The activity signal at 25°C will decrease approximately 3-4-fold relative to signal measured at 37°C.
• It is important to prepare all working solutions by thawing them briefly in a warm water bath and vortexing (except the enzyme solution). A quick centrifugation in a microfuge is recommended to limit loss of materials on tube sides and caps. Vortex and/or place the SDS solution briefly in a sonicating bath to ensure that it is in solution prior to use. Once thawed, the 20S proteasome enzyme solution should be place immediately on ice. SDS, substrate, and reaction buffer solutions can be stored at room temperature during the course of their use.
• Depending on what purpose this kit is being used for, the addition order of substrate, SDS, and 20S may vary. In all permutations listed below, components should be diluted into reaction buffer and mixed well prior to the addition of the kit reagents.

Table 1: Order of Addition of Reagents


• This assay kit is designed to be used with many types of agents to be tested in the assay. Although the concentration of SDS for maximal activation (0.03%) is below the critical micellar concentration (CMC), SDS still may interact with some agents, such as unstable proteins and highly positively charged agents. In such cases, an alternate method of activating the 20S proteasome is the activator PA28 (Cat. No. 506280).
• The 20S Proteasome solution as supplied is stable on ice for at least 24 h. Once activated with SDS, the activity is stable at 37°C for at least 1 h.
• Due to the inherent decreased sensitivity of fluorometric plate readers as compared to cuvette-based fluorimeters, when using the kit in plate format, higher enzyme concentration or longer incubation times with the substrate may be needed to produce a strong signal.
Detailed protocolCuvette Assay
1. Thaw all solutions briefly in a warm water bath. Vortex and centrifuge briefly. Make an appropriate amount of reaction buffer (diluted in H2O) for the number of samples planned and place the reaction buffer at 37°C (~1 ml per sample).
2. Pipette 985 µl of reaction buffer solution into a microcentrifuge tube.
3. Add 10 µl of 3% SDS solution and mix thoroughly.
4. Pipette 1 µl (~1 µg) of 20S proteasome into the 0.03% SDS solution tube, mix, and transfer the enzyme solution to the cuvette.
5. Allow 5 min for temperature equilibration.
6. Make a 200X (2 mM) Substrate Solution (1:5) diluted in reaction buffer. Pipette 5 µl of 200X Substrate Solution into the cuvette and mix. Monitor fluorescence signal (excitation max: ~380 nm; emission max.: ~460 nm) over time (e.g. measure the fluorescence every 10-20 s for 5-10 min).

Plate Assay
1. Thaw all solutions briefly in a warm water bath. Vortex and centrifuge briefly. Make an appropriate amount of reaction buffer (diluted in H2O) for the number of samples planned and place the reaction buffer at 37°C. Each well will require 200 µl total reaction volume.
2. Add SDS to the reaction buffer such that final concentration is 0.03% (~2 µl of 100X stock per sample). Add enzyme (0.2 µg/well) and incubate for 5 min for equilibration at assaying temperature.
3. Using a multi-channel pipette, aliquot 190 µl of the activated enzyme mixture into each well on the plate and allow for temperature equilibration.
4. Make a 20X (200 µM) Substrate Solution (1:50) diluted in reaction buffer. Each well will require 10 µl.
5. Initiate the reaction by adding 10 µl of the 20X Substrate Solution into each well.
6. Monitor fluorescence signal (excitation max: ~380 nm; emission max.: ~460 nm) over time.
Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
Interactive Pathways™ is a trademark of EMD Chemicals, Inc.