218693 Protease Substrate

218693
  
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      Casein labeled with N-(resorufin-4-carbonyl)piperidine-4-carbonic acid. Colorimetric or fluorometric substrate for sensitive detection of protease contamination in solution.
      Catalogue Number218693
      Brand Family Calbiochem®
      SynonymsCasein, Resorufin-Labeled
      References
      Product Information
      FormRed lyophilized solid
      Applications
      Biological Information
      Physicochemical Information
      Emission max.
      Excitation max.
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Ambient Temperature Only
      Toxicity Standard Handling
      Storage -20°C
      Do not freeze Ok to freeze
      Special InstructionsFollowing reconstitution, aliquot and freeze (-20°C) for long term storage or refrigerate (4°C) for short term storage. Stock solutions are stable for up to 3 days at 4°C and for up to 3 months at -20°C.
      Packaging Information
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      Specifications

      Documentation

      Protease Substrate MSDS

      Title

      Safety Data Sheet (SDS) 

      Protease Substrate Certificates of Analysis

      TitleLot Number
      218693
      Data Sheet

      Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.

      Revision03-June-2008 RFH
      SynonymsCasein, Resorufin-Labeled
      DescriptionCasein labeled with N-(resorufin-4-carbonyl)piperidine-4-carbonic acid. Colorimetric and fluorometric substrate for sensitive detection of protease contamination in solution.
      FormRed lyophilized solid
      Recommended reaction conditions
      Assay for Determination of Proteolytic Activity Note: This protocol is provided only as a general guide. Researchers should standardize this assay for their own specific needs and should consult published literature. Introduction: Protease substrate is milk casein that is coupled with activated resorufin (N-(resorufin-4-carbonyl)piperidine-4-carbonic acid N-hydroxysuccinimide ester). It is a general protease substrate and is particularly useful for the detection of trace protease activity. Treatment of the substrate with protease results in the release of resorufin-labeled peptides that are not precipitated by trichloroacetic acid (TCA). The concentration of the labeled peptides in the supernatant is proportional to the proteolytic activity in the sample. The following protocol has been modified according to Twining, S.S. Reagents and Equipment: Sample solution Substrate solution: 0.4% protease substrate (casein, resorufin-labeled, Cat. No. 218693) in dH2O Incubation buffer: 0.2 M Tris-HCl, pH 7.8, 0.02 M CaCl2 Stop buffer: 5% Trichloroacetic acid (w/v) in dH2O Assay buffer: 0.5 M Tris-HCl, pH 8.8 Spectrophotometer capable of measuring absorbance at 574 nm (colorimetric) or fluorimeter capable of measuring excitation at 574 nm and emission at 584 nm (fluorometric) Incubator capable of maintaining constant temperature at 37°C Protocol: For each sample, mix the following: 50 µl substrate solution 50 µl incubation buffer 100 µl sample solution Prepare a blank by mixing the following: 50 µl substrate solution 50 µl incubation buffer 100 µl dH2O Incubate samples and blank at 37°C for the desired amount of time (15 min to overnight) Note: The incubation time will depend on the amount of protease present in the unknown sample(s). It may be necessary to perform a time-course study to determine the optimal incubation time for a given sample. Various concentrations of protease have yielded the following results: Concentration        Color Change Within 2 mg/ml                         ~1 min. 0.5 mg/ml                      ~5 min. 0.2 mg/ml                      ~10 min. Stop the reactions by adding 480 µl of stop buffer (samples and blank) Incubate samples and blank at 37°C for 10 min Centrifuge for 5 min and transfer 400 µl of each supernatant to a cuvette Add 600 µl of assay buffer, mix, and immediately read the absorbance of the sample against the blank at 574 nm, 15-25°C. The color will change from a bluish-violet to red. Alternatively, the sensitivity may be improved by measuring the cleavage in a fluorimeter with an excitation wavelength of 574 nm and an emission wavelength of 584 nm
      SolubilityH₂O (4 mg/ml)
      Storage -20°C
      Do Not Freeze Ok to freeze
      Special InstructionsFollowing reconstitution, aliquot and freeze (-20°C) for long term storage or refrigerate (4°C) for short term storage. Stock solutions are stable for up to 3 days at 4°C and for up to 3 months at -20°C.
      Toxicity Standard Handling