Introduction

This document describes how to use an Odyssey M Imager and LI‑COR® Acquisition Software to acquire an image of a protein concentration estimation assay performed in a multiwell plate. Empiria Studio® Software will be used for analysis.

The following protein estimation assays are described in this document.

This guide covers image acquisition and analysis. Instructions for how to use a protein concentration estimation kit will vary depending on the type of assay and the manufacturer of the kit. Follow instructions provided by the manufacturer.

Brief Background About Protein Concentration Estimation

Estimating protein concentration is essential for a reliable Western blot assay. Only by estimating protein concentration can you be sure how much protein you are loading into the gel.

Planning the Experiment

Please look to the instructions for the protein estimation kit (or other materials) that you are using while planning your experiment. This document focuses on analysis, not experimental planning or lab protocol.

Be aware that the concept called a "blank" in many protein estimation kit instructions is covered by the term "background" in Empiria Studio. The word "background" is used instead of "blank" to accommodate similar concepts across the wide variety of assays that can be analyzed in Empiria Studio.

Required Materials

The following materials are required to follow the instructions in this document.

• Odyssey M Imager

  The Odyssey M Imager provides three transillumination channels that are used to image absorbance-based assays: 470 Trans, 525 Trans, and 630 Trans. See licorbio.com/DetectionLabelReference for a complete list of Odyssey M Imager detection channels. See the Odyssey M Imager manual (licorbio.com/m-manual) for more information about the differences between trans illumination, epi illumination, and fluorescence detection.

• Multiwell plates

  Clear plates and black or white plates with clear bottom plates have been used for absorbance-based assays on the Odyssey M Imager. Clear plates tend to be recommended for absorbance-based assays and will provide an image with the expected visual representation of the data.

• LI‑COR Acquisition Software version 1.0 or version 1.0.1 (licorbio.com/AcquisitionQuickStart)

• Empiria Studio® Software version 2.0, version 2.1, or version 2.1.1 (licorbio.com/empiria)

• Materials (such as from a kit) for performing one of the following assays: Micro BCA Assay, Bradford (Coomassie Plus) Assay, Pierce 660 Assay, Modified Lowry Assay

  Follow the manufacturer's instructions for your kit or for individual materials you will be using to prepare the assay.

Place Multiwell Plates on the Odyssey M Imager

Place up to four multiwell plates on the Odyssey M Scan Surface. Use the Plate Alignment Guide (926-18972) to align plates on the Scan Surface.

Acquire Image

Follow the steps below, depending on which type of protein estimation assay you want to image.

Acquire Micro BCA Assay Image

1. Start LI‑COR® Acquisition Software .

2. Connect to the Odyssey M Imager.

3. On the Choose Assay page, click Custom.

4. In the Assay list, click Absorbance Assay.

5. Select the locations on the Scan Surface that have a multiwell plate to image.

6. Click Next.

7. In the Select Detection Channel dialog, select 525 Trans and click Save.

   Do not select any other channel. Selecting only 525 Trans is essential for analysis in Empiria Studio.

8. On the Detection Options page, select the following options.

   • Select the 96 Well well format option. The 384 Well option has not been evaluated for protein estimation assays.

   • Leave the default focus offset (3.00 mm). Absorbance detection is not sensitive to the focus offset value.

   • Leave the default resolution (100 µm).

9. Click Scan.

10. When the image has been acquired, click Export to Analyze.

    This option will export a Zip File. The Zip File can be imported into Empiria Studio for analysis.

11. When you are done, click Leave.

Acquire Other Protein Estimation Assay Images

Follow these instructions to acquire an image for the following types of protein concentration estimation assays: Bradford (Coomassie Plus) Assay, Pierce 660 Assay, Modified Lowry Assay.

1. Start LI‑COR® Acquisition Software .

2. Connect to the Odyssey M Imager.

3. On the Choose Assay page, click Multiwell Plate.

4. On the Choose Multiwell Plate Assay page, click Absorbance Assay.

5. Select the locations on the Scan Surface that have a multiwell plate to image.

6. Click Next.

7. In the Select Detection Channel dialog, select 630 Trans and click Save.

8. On the Detection Options page, select the following options.

   • Select the 96 Well well format option. The 384 Well option has not been evaluated for protein estimation assays.

   • Leave the default focus offset (3.00 mm). Absorbance detection is not sensitive to the focus offset value.

9. Click Scan.

10. When the image has been acquired, click Export to Analyze.

    This option will export a Zip File. The Zip File can be imported into Empiria Studio for analysis.

11. When you are done, click Leave.

Analyze Protein Concentration Estimation Assay in Empiria Studio® Software

To analyze a protein estimation assay in Empiria Studio, follow the instructions in this section.

Import Zip File for Analysis

1. Start Empiria Studio .

2. Click Image Gallery .

3. Click Import LI-COR Zip Files, and import the Zip File you exported from LI‑COR Acquisition Software.

Create Plate Template

If you already have a Plate Template that can be used to analyze your protein estimation plate image, you do not need to repeat these steps. You can skip to Analyze Plate.

Plate Templates are used to analyze multiwell plate images in Empiria Studio. In the Plate Template, you indicate which wells are used and what the wells contain. During analysis, you choose a Plate Template and an image. The image is quantified and calculations are performed automatically based on the information you specified in the Plate Template.

You can reuse Plate Templates. You may only need to create one Plate Template for analyzing protein concentration estimation assays. The Plate Template for protein estimation assays can be very simple, because only the Total pixel intensity is needed.

Follow these steps to create Plate Template for analyzing a protein estimation assay:

1. On the Empiria Studio Home page , click Template Library & Experiment Designer .

2. Click Create Template.

3. On the Add Template Details page, enter the following information.

   • Enter a name for the Plate Template.

   • In the Assay list, choose Cell Analysis. When the Category options appear, choose Target Analysis.

   • In the Well Format list, choose 96 Well Plate.

4. Click Next.

5. On the Set Backgrounds page, select all the wells to use for background and click Set as Background.

   The word "background" is used instead of "blank" to accommodate the wide variety of assays that can be analyzed in Empiria Studio.

6. Click Next.

7. Click Next until you are on the Set Samples page.

8. On the Set Samples page, click and drag to select all the wells you want to analyze. Click Set as Sample.

9. Click Next.

10. On the Set Well Groups page, click All Wells.

11. Click Next.

12. On the Review page, click Done.

Analyze Plate

If you do not have a Plate Template that can be used for protein estimation assay analysis, refer to Create Plate Template before following these steps.

1. On the Empiria Studio Home page , click Projects .

   You can use an existing Project or create a new one.

2. Click Create New Experiment.

3. Click Multiwell Plate.

4. Click Absorbance Assay.

5. Click Target Analysis.

6. Click Image Gallery, and select the image Acquisition you want to analyze.

7. Click Start.

8. On the Name page, enter a name.

9. Click Next.

10. On the Select Template page, click Select. Select the Plate Template you want to use and click Select. See Create Plate Template for more information.

11. Click Next.

12. On the Quantify wells page, adjust the plate boundary, if needed.

    • Click Analysis Table to view well quantification values.

    • Click to export the quantification value data to Excel. Name the file and choose the location to save the Excel file.

Perform the remainder of the analysis steps outside of Empiria Studio. For example, fit the standards using appropriate regression analysis (e.g., linear or 4PL regression) and interpolate unknown sample concentrations.