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 may be able to bind PF3D7_1351100, the slides were imaged using a Genepix microarray scanner. The scanner measured the fluorescence signal emitted from the slide at two wavelengths: 532 nm
and 635 nm. The goal for today is to familiarize you with how the SMM slides are scanned and imaged. These images are the raw data that will be used to identify putative small molecule binders.
Overview of SMM slide imaging.
A. To visualize which small molecule is putative binder of PF3D7_1351100, the 6xHis-tag is labeled using an anti-His antibody that is conjugated to a fluorophore. B. The fluorophore used to label protein bound to the small molecule in a particular location on the slide emits a red signal (indicated by white arrow). Image in Panel A generated using BioRender.
When the SMM slides are imaged, the scanner exposes each slide to excitation light specific to the fluorophores used in the experiment. As shown in the figure above, two fluorophores were used to evaluate small molecule binding to PF2D7_1351100. The green spots represent locations on the SMM slide where fluorescein was printed. Fluorescein is a fluorescent dye that emits at 532 nm and is used for alignment purposes. Correct alignment is critical to knowing which small molecules are in which spots on the slide. Red spots are indicative of small molecules that are bound by PF3D7_1351100. The signal is due to an Alexa Fluor 647 anti-His antibody that emits at 635 nm.
During imaging, the scanner detects the intensity of the emitted fluorescence light to generate an image that can be analyzed. The intensity and position of the 'red' signal associated with putative small molecule 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 to identify drug candidates.
Part 1: Participate in Comm Lab workshop
Our communication instructors, Dr. Prerna Bhargava and Dr. Sean Clarke, will join us today for a discussion on preparing a Journal club presentation.
Part 2: Scan SMM slides
To ensure you are familiar with the steps involved in imaging the SMM experiment, please watch the video tutorial provided by a researcher from the Koehler Laboratory linked here: SMM Scanning. The steps are detailed below so you can follow along!
- Place the slide in the scanner barcode side down.
- Set the desired wavelengths that you want to scan.
- For fluorescein: 532 nm
- For Alexa Fluor 647: 635 nm
- Set the wavelength, filter, PMT, and power settings.
- Run a preview scan to optimize the PMT for the 635 nm (red) emission.
- Complete a full scan using the optimal PMT value.
- Following the scan, you will see an image of your slide as below.
In your laboratory notebook, complete the following:
- What color is emitted by fluorescein? What does this indicate on the scan (what is present in spots that emit this signal)?
- What color is emitted by Alexa Fluor 647? What does this indicate on the scan (what is present in spots that emit this signal)?
Part 3: Review journal article
Critically a and discuss the following journal article with your laboratory partner:
Amberg-Johnson et al. "Small molecule inhibition of apicomplexan FtsH1 disrupts plastid biogenesis in human pathogens." eLife. (2017) 6:e29865.
The initial experiment presented by Amberg-Johnson et. al. shows the effect of actinonin on apicoplast biogenesis. The apicoplast is an essential plastid organ that is a key target for drug development in research focused on malaria treatment. Actinonin was identified in large-scale screen of compounds known to inhibit growth of parasite. The subsequent experiments completed in this research served to uncover the mechanism-of-action of actinonin is it pertains to disruption of the apicoplast.
In the context of your research, this article focuses on the next step experiments that can be performed after a drug candidate is discovered from a screen. Though you can use this article as guidance as you consider the experiments that could follow your screen, remember that the specific next step experiments should be related to the protein target and drug candidate(s) identified in your project. For this exercise, the focus in on how the data are organized and presented.
In your laboratory notebook, complete the following with your partner:
- Why is the apicoplast a promising target for anti-malarial drug development?
- Why have attempts at developing broadly effective drugs that target the apicoplast been unsuccessful?
- Why is the approach used by the researchers in this article more promising?
- List the figures that are included in the article. For each figure:
- What is the main conclusion / finding in each figure?
- Which panel best supports the main conclusion / finding? Is more than one panel needed to fully support the main conclusion?
- Are you convinced by the data? Do you agree with the main conclusion?
- Are the figures organized in a coherent story?
- Write transition statement that connect each figure to the next. A transition statement should very briefly summarize the findings of a figure and state what those findings motivated the research to do next (ie what is the next experiment?).
Part 4: Edit homework assignment
Using the insight gained from Part 3 and the pointers from the Comm Lab workshop, edit the slide you prepared for the homework assignment due today. You can submit the updated slide by 10 pm tonight for grading. You will get a grade for submitting the original slide on-time and the updated slide will be graded for content. If you choose not to update your homework, the slide already submitted will be graded for content.
- Genepix 4300 microarray scanner (Molecular Devices)
- Genepix Pro software (Molecular Devices)
Next day: Analyze SMM data to confirm putative small molecule binders
Previous day: Prepare small molecule microarray (SMM) slides with purified protein