20.109(F20):M2D5

Introduction

In the previous laboratory session the printed SMM slides were incubated with the purified PF3D7_1351100 protein in an effort to identify putative small molecule binders. To check which of the small molecules, if any, you screened are able to putatively bind PF3D7_1351100, the slides were evaluated using a Genepix microarray scanner. The scanner measured the fluorescence signal emitted from each spot on the slide where a ligand was printed and generated an image of each slide that will be analyzed in the next laboratory session. The goal for today is to familiarize you with how the plates are scanned and imaged. These images are the raw data that will be used to identify putative small molecule binders.

UDPATE IMAGE

The above image (right panel) is from a pilot experiment completed in preparation for this module. The green spots represent locations on the SMM slide where fluorescein was printed. As a reminder, fluorescein is a fluorescent dye used for alignment purposes. Correct alignment is critical to knowing which ligands are in which spots on the slide. Red spots are indicative of ligands that are bound by PF3D7_1351100 (example highlighted by white arrow in right panel). The signal is due to ANTI HIS ANTIBODY??? (schematic shown in left panel).

When the SMM slides are imaged, the scanner exposes each slide to excitation light specific to the fluorophores used in the experiment (i.e. wavelengths that excite fluorescein and the ????? attached to PF3D7_1351100). The scanner then detects the intensity of the emitted fluorescence light to generate an image that can be analyzed. The intensity and position of the signal associated with putative ligand binders is what will be assessed in the next laboratory session. This analysis will provide a list of 'hits' that can be used in future studies.

Protocols

Part 1: Scan slides

A researcher from Prof. Koehler's Laboratory will lead you through a demonstration on how to scan your SMM slides. You should take notes to ensure you know the purpose of each setting. An outline of the steps is included below for your reference.

GET A VIDEO????

1. Place the slide in the scanner barcode side down.
2. Set the desired wavelengths that you want to scan.
   • For fluorescein: 532 nm
   • For Alexa Fluor 647: 635 nm
3. Set the wavelength, filter, PMT, and power settings.
   • The Koehler Lab member will discuss these features during the demonstration.
   • Record the purpose for each of these settings in your laboratory notebook!
4. Run a preview scan to optimize the PMT for the 635 nm (red) emission.
5. Complete a full scan using the optimal PMT value.
6. Following the scan, you will see an image of your slide as below.

Part 2: Review journal article

Skim and discuss the journal article by Chen et al. titled "Small molecule microarrays enable the discovery of compounds that bind the Alzheimer's αβ peptide" with your laboratory partner.

The initial experiment presented within this publication was an SMM screen that identified small molecule binders of the αβ petptide. This first step is very similar to what you completed in this module! To further assess the results of the SMM, Chen et al. completed several follow-up experiments.

In your laboratory notebook, complete the following with your partner:

• List the experiments that were reported by the researchers and the conclusions of these experiments. You do not need to provide much detail here, just a sentence or two with what was done and what information/insight was gained.
• Consider how these experiments are connected such that the paper provides a complete and coherent story. What is the story?

In your laboratory notebook, complete the following individually:

• Use this list generated with your partner as a guide to organize the data that you will include in your Research article. To get you started, create an outline that orders the figures that will be included in the assignment and record your thoughts on how the figures relate to each other (ie what is your story and how do the data support your story?).
• Think about follow-up experiments that would be useful / insightful if you were to assess the putative binders identified in the SMM screen for PF3D7. How might you introduce / organize the follow-up experiments in the Implications and Future works section of your Research article? **For ideas on future works you can consider the follow-up experiments completed by Chen et at.; however, keep in mind that not all of the approaches in the paper are relevant to your research! The questions that you address in your future works experiments should work toward a complete and coherent story.

Reagents

• Genepix 4300 microarray scanner (Molecular Devices)
• Genepix Pro software (Molecular Devices)

Navigation links

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