488250 Non-Interfering Protein Assay™ Kit

488250
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488250-1KTT
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      Description
      OverviewAn easy-to-use protein assay that overcomes interference of agents found in protein solutions including detergents, chelating agents, reducing agents, amines, sugars, urea, and others. The Universal Protein Precipitating Agent (UPPA™ Reagent) is used to precipitate and immobilize the protein in the tube while the interfering reagents are removed. Protein concentration is based on the specific binding of copper to the peptide backbone. As the protein concentration increases, the concentration of unbound copper ions decreases, and the color density is inversely related to the amount of protein present in solution.
      Catalogue Number488250
      Brand Family Calbiochem®
      Application Data

      Materials Required but Not Delivered 2 ml tubes
      References
      ReferencesHo, T.H. et al. 2005. Human Molecular Genetics 14, 1539.
      Ladd, A.N. et al. 2001. Mol. Cell. Biol. 21, 1285.
      Loeb, D.M. et al. 2002. J. Biol. Chem. 277, 19627.
      Shiels, A. et al. 2007. Invest. Ophthalmol. Vis. Sci. 48, 500.
      Gushwa, N.N. et al. 2003. Plant Physiology 132, 1925.
      DePinto, W. et al. 2006. Mol. Cancer Ther. 5, 2644
      Werner, M.E. et al. 2007. J. Biol. Chem. 282, 5560.
      Dennison, S.M. et al (2006). Biophysical Journal 90, 1661.
      Reeve, I. et al. 2002. PNAS 99, 8608.
      Product Information
      Detection methodColorimetric
      Form500 Tests
      FormatCuvette or 2 ml, 96-deep-well plate
      Kit containsUPPA™ Reagents I and II, Copper Solution I, Color Agents A and B, BSA Standard, and a user protocol.
      Applications
      Biological Information
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 36/37/38

      Irritating to eyes, respiratory system and skin.
      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
      Intended useThe Non-Interfering Protein Assay™ is a highly sensitive colorimetric assay that overcomes interference by common laboratory agents. The assay removes detergents (non-ionic, ionic and zwitterionic), reducing agents (β-mercaptoethanol, DTT), chelating agents (EDTA), amines (Tris), sugars, and is highly tolerant of strong chaotropic buffers. It is suitable for determining protein concentrations in protein loading buffer (Laemmeli buffer), high β-mercaptoethanol concentrations (<15%), and in lipid and vesicle preparations. The Non-Interfering Protein Assay™ is linear between 0.5-50 µg and requires a small sample (1-50 µl).
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage +15°C to +30°C
      Storage ConditionsUpon arrival, store UPPA-I and UPPA-II at room temperature and the remaining components at 4°C, in the dark in the original box.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsUPPA™ Reagents I and II, Copper Solution I, Color Agents A and B, BSA Standard, and a user protocol.
      Specifications

      Documentation

      Non-Interfering Protein Assay™ Kit Certificates of Analysis

      TitleLot Number
      488250

      References

      Reference overview
      Ho, T.H. et al. 2005. Human Molecular Genetics 14, 1539.
      Ladd, A.N. et al. 2001. Mol. Cell. Biol. 21, 1285.
      Loeb, D.M. et al. 2002. J. Biol. Chem. 277, 19627.
      Shiels, A. et al. 2007. Invest. Ophthalmol. Vis. Sci. 48, 500.
      Gushwa, N.N. et al. 2003. Plant Physiology 132, 1925.
      DePinto, W. et al. 2006. Mol. Cancer Ther. 5, 2644
      Werner, M.E. et al. 2007. J. Biol. Chem. 282, 5560.
      Dennison, S.M. et al (2006). Biophysical Journal 90, 1661.
      Reeve, I. et al. 2002. PNAS 99, 8608.
      User Protocol

      Revision01-November-2010 JSW
      Form500 Tests
      FormatCuvette or 2 ml, 96-deep-well plate
      Detection methodColorimetric
      StorageUpon arrival, store UPPA-I and UPPA-II at room temperature and the remaining components at 4°C, in the dark in the original box.
      Intended useThe Non-Interfering Protein Assay™ is a highly sensitive colorimetric assay that overcomes interference by common laboratory agents. The assay removes detergents (non-ionic, ionic and zwitterionic), reducing agents (β-mercaptoethanol, DTT), chelating agents (EDTA), amines (Tris), sugars, and is highly tolerant of strong chaotropic buffers. It is suitable for determining protein concentrations in protein loading buffer (Laemmeli buffer), high β-mercaptoethanol concentrations (<15%), and in lipid and vesicle preparations. The Non-Interfering Protein Assay™ is linear between 0.5-50 µg and requires a small sample (1-50 µl).
      Principles of the assayThe Non-Interfering Protein Assay™ is composed of two simple steps. First, the Universal Protein Precipitating Agent (UPPA™) is added to the protein solutions to rapidly precipitate total protein. The protein is immobilized by centrifugation and interfering agents in the supernatant are discarded. Second, the protein concentration is assayed by mixing the sample with an alkaline solution containing a known concentration of copper salt; the copper ions bind to the peptide backbone and the assay measures the unbound copper ions. The assay is independent of protein side chains minimizing protein-to-protein variation. The absorbance is inversely proportional to the amount of protein in the sample.




      Figure 1: Non-Interfering™ Protein Assay Overview

      Materials providedThe kit is supplied with enough reagents for 500 assays using Universal Protein Precipitating Agent (UPPA).

      • UPPA-I (Kit Component No. KP15001-250ML): 1 bottle, 250 ml
      • UPPA-II (Kit Component No. KP15002-250ML): 1 bottle, 250 ml
      • Copper Solution-Reagent-I (Kit Component No. KP15003-50ML): 1 bottle, 50 ml
      • Color Agent-A (Kit Component No. KP15004-250ML): 2 bottles, 250 ml each
      • Color Agent-B (Kit Component No. KP15005-5ML): 1 vial, 5 ml
      • Protein Standard BSA (2 mg/ml) (Kit Component No. KP15006-5ML): 1 vial, 5 ml
      Materials Required but not provided 2 ml tubes
      Precautions and recommendations Always use clean, sterile pipets and aseptic techniques for removing reagents from the reagent bottles.
      Reagent preparation• Reagent II: Prior to use, prepare an appropriate volume of Reagent II by mixing 100 parts Color Agent-A with 1 part Color Agent-B. Mark this solution as Reagent II. Fresh or unused Reagent II can be stored at 4°C and is stable for up to 1 month or as long as the absorbance of the solution remains below 0.025 at 480 nm.
      Detailed protocolPerform the assay at room temperature.
      1. Prepare a set of protein standards using the supplied BSA Protein Standard as indicated in the table below:

      Table 1: Set of Protein Standards


      2. Add 1-50 µl of unknown protein samples to 2 ml tubes.

      NOTE: It is recommended that samples be assayed in duplicate. The total amount of protein should not exceed 50 µg, so it is recommended that several dilutions of samples be assayed to ensure that samples are below 50 µg.
      NOTE: For determination of protein concentrations in buffers free of interfering agents skip steps 3-6 (i.e., do not add UPPA-I or UPPA-II if your buffer does not contain any interfering agents).

      3. Add 500 µl UPPA™ I to each tube and vortex. Incubate for 2-3 min at room temperature.
      4. Add 500 µl UPPA™ II to the tubes and vortex.
      5. Centrifuge the tubes at maximum speed (~10,000 x g) for 5 min to pellet the precipitated protein. For easier identification of the pellet, ensure all the tubes are centrifuged with the cap hinge facing outwards. A small pellet should be visible.
      6. Decant the supernatant, return the tubes to the centrifuge, pulse to centrifuge the residual liquid, and remove with a pipette. OPTIONAL: For enhanced washing for problematic samples see the Troubleshooting section.
      7. Add 100 µl Copper Solution (Reagent I) and 400 µl deionized water to the tubes and vortex until the protein precipitate pellet dissolves.
      8. Using a 1-ml pipette, rapidly shoot 1 ml Reagent II directly into each tube containing Copper Solution (Reagent I) plus diH2O and immediately mix by inverting the tubes.
      11. Incubate at room temperature for 15-20 min and then immediately read absorbances at 480 nm against diH2O.
      10. Plot the absorbance against the protein concentration and determine protein concentrations of unknowns by comparing to the standard curve. NOTE: Do not subtract blank reading from the sample reading as absorbance will decrease as protein concentration increases.

      Protocol For High Throughput 96-Well Assays

      NOTE:
      For high throughput 96-well assays, we recommend using 2 ml deep round or V- bottom well titer plates. The high throughput protocol requires centrifugation of the 96-well plate at 2-5000 x g and this may require a special centrifuge adaptor.

      1. For performing the high throughput, in a 96-well format, follow steps 1-5 of the above protocol.
      2. Centrifuge the plate at ~5000 x g for 7 min to pellet the precipitate. Invert the plate to remove the supernatant and shake to remove all excess supernatant.
      3. Continue with the above protocol following steps 8-10.
      4. After incubation, transfer 200 µl assay reaction to a flat bottom 96-well plate and measure the absorbance at 480 nm against DI water.
      5. Plot the absorbance against protein concentration and determine protein concentrations of unknowns.
      NOTE: Do not subtract blank reading from the sample reading as absorbance will decrease as protein concentration increases.
      Limitations of the assay

      Table 2: Tolerance Guide









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