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Pierce 660 NanoDrop Microplate protocol.pub


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									                                                      PROTOCOL                                NanoDrop 1000  &  NanoDrop 8000

                                                      Pierce 660 nm Protein Assay
    Introduction                                                              Sample Preparation
    The Thermo Scientific Pierce 660 nm Protein Assay reagent                 1. Equilibrate all reagents, unknowns and protein standards to
    is a ready-to-use formulation that offers rapid, accurate and             room temperature.
    reproducible colorimetric detection of minute amounts of
    protein in solution. Used in conjunction with the micro-                  2. Mix each stock standard solution and unknown sample
    volume capability of the Thermo Scientific NanoDropTM                     gently prior to use.
    Spectrophotometers, the reagent provides an accurate and
    rapid means of protein quantitation with minimal                          3. Prepare a zero reference (0 mg/ml protein) by adding 10 of
    consumption of sample.          The ability of NanoDrop                   the assay buffer to 150 ul of the Pierce 660 reagent.
    spectrophotometers to measure as little as 2 ul of protein
                                                                              Note: Whether using a predefined standard curve or
    samples allows significantly scaled-down reaction volumes,
                                                                              generating a new curve, the zero reference solution is used as
    thereby using only a fraction of sample and reagent
                                                                              the ‘blank’. This is unlike the other colorimetric assays run on
    commonly needed for conventional cuvette-based
                                                                              NanoDrop instruments where water is used for the ‘blank’
    Dynamic Range                                                             4. Transfer 10 ul of each sample or standard to 150 ul of the
    The assay has a linear range of 50-2000 ug/ml using a                     Pierce 660 reagent.
    sample to reagent ratio of 1:15. The sensitivity of the assay
    may be increased by using a 1:7.5 sample to reagent ratio                 5. Mix each standard and unknown sample thoroughly by
    yielding a linear range of 25-1000 ug/ml.                                 gently pipetting up and down several times.

                                                                              6. Collect the solution at the bottom of the tube by a brief
    Supplies                                                                  centrifugation.
    − NanoDrop 1000 or NanoDrop 8000                                          7. Incubate at room temperature for 5 minutes.
    − 1-10 ul pipettor (low retention tips)                                   Example spectrum of a Pierce 660 nm Protein sample.
    Materials:                                                                Note: The assay is read at 660 nm due to better sensitivity at
    − Low lint laboratory wipes                                               lower concentrations.
    − 0.5 ml Eppendorf tubes or 0.2 ml mini-centrifuge
      strip tubes and caps

    − Pierce 660 nm Reagent, Pierce Product # 22660
    − Pierce pre-diluted BSA standards Pierce Product
      #23208(OPTIONAL)(or other protein standard)

    Assay Recommendations
    − Use 2 ul aliquots for all measurements
    − Mix all solutions thoroughly but gently to avoid
         micro bubbles.
    − For additional information regarding the Pierce 660nm
       reagent, please refer to the Pierce Website.

Thermo Fisher Scientific - NanoDrop products   Wilmington, Delaware USA   Technical support: info@nanodrop.com   302-479-7707     www.nanodrop.com
             2                                                  PROTOCOL                                NanoDrop 1000  &  NanoDrop 8000

   Standard Curve and Sample Measurements
   Schematic of standard curve options for the NanoDrop 1000                                   6. Under the Measurement type– Standards tab- enter
   and 8000:                                                                                      the values for up to 7 standards.

                                                                                               7. Click on the Standards tab. Highlight the Reference
                                                                                               8. Mix the reference solution briefly and transfer 2 uL
             Load Predefined                           Build Your Own
                 Curve                                      Curve
                                                                                                  of the solution onto the lower pedestal. Lower the
                                                                                                  arm and click the Measure button (F1).
             Choose Sample                        Enter standard values                        9. Measure up to 5 replicates of the reference solution
                 Format                                                                           using a fresh 2 uL aliquot for each measurement.
                                                                                                  Repeat this step for each standard .
          Blank on Reference                          Blank on Reference
                                                                                               10. Once the standard curve is completed, select the
                                                                                                  Standard Curve Type (Interpolation, Linear, 2°
            Measure samples                           Measure standards                           polynomial, 3° polynomial) that best fits the
                                                                                                  standards data set.

                                                       Choose Sample                                Example standards dilution series:

                                                      Measure samples                                      BSA             A660
                                                                                                        (ug/ml)           (n=5)        St dev     %CV
                                                                                                            0                0           NA       NA
                                                                                                          125              .039         .008       1.6
   1. Clean upper and lower pedestals with 2 uL of dH20.                                                  250              .077        .0008       0.9
   2. From the Main Menu, open the Protein Pierce 660                                                     500              .158        .0026       1.5
      module.                                                                                             750              .229        .0013       0.5
                                                                                                          1000             .303        .0016       0.5
   3. Establish a blank using the appropriate buffer. Pipette 2 ul
      of the zero reference solution onto the bottom pedestal,                                 11. Click on the Sample tab under Measurement Type,
      lower the arm and click the “Blank” button.                                                  and enter the unknown samples’ respective ID
   4. Wipe the upper and lower pedestals with a dry laboratory                                     information. If a dilution of the unknown sample
      tissue. Repeat this step after each measurement.                                             was made, enter the dilution factor in the box
                                                                                                   below the sample ID window.
   5. Generate or load a standard curves. Options include:
                                                                                                     Note: If using the NanoDrop 8000, choices for
   − Load Standard Curve- load standard curves with                                                  entering sample ID information include loading a
     predefined concentrations and corresponding values.                                             pre-formatted plate file, entering sample
     After loading the curve– proceed to step 11.                                                    information with a bar code reader or manually
                                                                                                     inputting the sample IDs for each unknown sample.
   − Manual Curve Entry- generate absorbance values for a
     predefined standard concentration series. After loading                                   12. Mix each sample briefly prior to measurement and
     the concentration series, proceed to step 7.                                                  transfer 2 uL onto the lower pedestal (s).

   − New Standard Curve- generate new curves with user                                         13. Raise the sampling arm and wipe the upper and
     defined standard concentrations. Proceed to step 6.                                           lower pedestals with a laboratory tissue. Repeat this
                                                                                                   step after each measurement.
                                                                                                                                                         Rev 7/08
Thermo Fisher Scientific - NanoDrop products            Wilmington, Delaware USA   Technical support: info@nanodrop.com           302-479-7707   www.nanodrop.com

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