Recursos Técnicos - Notas Técnicas
In-situ Micro-Volume BCA Assay - Using Epoch™ & Take3™ Micro-Volume PlateDescargar
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.
- Bicinchoninic Acid Protein Assay Kit (Sigma, PN-BCA1 and B9643)
- Purified water (like MilliQTM)
- Single- and 8-channel pipettor with 1-10 μL range
- 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:
- Select the Take3 plate from the Plate Type dropdown
- Read step: 562 nm, Normal speed, wells A2->H3
- The example Plate Layout is shown below, using the well locations A2-H3, corresponding to the microspots of the Take3 plate:
- 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