We use cookies to provide visitors of our website with the best possible experience. To learn more how we use cookies or how to block cookies, please visit our cookie policy.

Resources - Technical Notes

In-situ Micro-Volume BCA Assay - Using Epoch™ & Take3™ Micro-Volume Plate


Related Products: Epoch, Gen5 for Detection

July 22, 2010


This Tech Note describes the materials, methods and Gen5™ parameters used to perform an in-situ microvolume based bicinchoninic acid (BCA) assay using the Take3 plate and Epoch reader. For more general information regarding Gen5™ Data Analysis Software (e.g. general concepts, data analysis, etc.) please refer to the software’s help system. Be sure to thoroughly read the application note titled In-situ Micro-Volume Bicinchoninic Acid Protein Assay on www.biotek.com.


Materials Used:

  • Bicinchoninic Acid Protein Assay Kit (Sigma, PN-BCA1 and B9643)
  • Purified water (like MilliQTM)
  • Single- and 8-channel pipettor with 1-10 μL range


Assay Setup:

  • The BCA Working Reagent was prepared just prior to use as per the manufacturer’s recommendations.
  • A 6 point 1:3 serial dilution series of protein standards was creating from a stock solution of BSA at 1 mg/mL (Sigma, PN-3294) in MilliQTM water. First, 2 μL of protein standards and samples were sequentially loaded directly onto the Take3 microspots, followed by 2 μL of BCA Working Reagent (using an 8-channel pipettor with mixing).
  • The blanks were made of a 1:1 volume ratio BCA working reagent and MilliQTM water.
  • The reaction was incubated at room temperature (~22ºC) for 25 minutes then read on the EpochTM Microplate Reader.


Epoch & Gen5/Take3 Setup:

  • Ensure that the Take3 plate is defined in the Gen5 Take3 Module
  • With the Take3 plate defined, create a standard Gen5 protocol to run this assay. See details below.


Gen5 Recommended Protocol Setup:

  1. Select the Take3 plate from the Plate Type dropdown
  2. Read step: 562 nm, Normal speed, wells A2->H3
  3. The example Plate Layout is shown below, using the well locations A2-H3, corresponding to the microspots of the Take3 plate:

    plate layout
  4. Data Reduction steps required:

    Data Reduction steps required:
    4a. Create a transformation to calculate pathlength corrected ODs to a 0.5 mm equivalent, using the pathlengths given on the Take3 data sheet.
    4b. Create a transformation to blank the pathlength corrected ODs, using the “0” STDs as the blanks.
    4c. Create a curve step:
    - “blanked A 562” is selected for the y-axis
    - use the 2nd degree polynomial curve fit