Atomic Force Microscopy to Visualize Phage–Antigen Interactions

תקציר

Source: Williams, S. M. et al. Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis. J. Vis. Exp. (2015)

This video demonstrates tapping-mode AFM to visualize phage–antigen interactions on a mica surface. It outlines the steps for antigen immobilization, phage binding, and high-resolution nanoscale imaging.

פרוטוקול

1. Atomic Force Microscopy (AFM) Process

1. Add 10-20 μl of the target antigen to cleaved mica and incubate for 5-10 min. Wash the mica 5 times with water. Add 10-20 μl of the phages to the mica and incubate for 5-10 min.
2. Wash the mica 5 times again with water and allow the mica to air dry. Visualize phage binding using AFM. Execute the AFM imaging process using AFM tapping mode with a Nanoscope IIIa controller in air at room temperature (RT). The silicon AFM probes have a resonant frequency of 300 kHz and a spring constant of 40 N/m. Use a scan rate of 3.05 Hz and a scan resolution of 512 samples/line.
3. Process the images by basic flattening of the background to ensure that all particle sizes are comparable within each image. Phage width (not length) may vary from image to image due to other particle sizes and the scanning area, but it will never compromise the accuracy of whether or not the phage binds to the antigen.
4. If during AFM verification for each set of negative panning, phages are visible on the mica, perform additional rounds until no phages are detected before proceeding to the next set of negative panning.

חומרים

רשימת החומרים שנעשה בהם שימוש במאמר זה

שם	חברה	מספר קטלוג	הערות
Tomlinson I and J Libraries	MRC (Cambridge, England)
Sheets Library	MRC (Cambridge, England)
KM13 Helper Phage	MRC (Cambridge, England)
Sodium Chloride	Macron	7647-14-5
Sodium Phosphate Dibasic	Amresco	0404-1KG	Irritant
Potassium Chloride	EMD	PX1405-1	Irritant
Potassium Phosphate Monobasic	Amresco	0781-500G	Irritant
Mica	Spruce Pine Mica	24365
Centrifuge	Thermo Scientific	Sorvall RC 6+
Nanoscope IIIa Atomic Force Microscope	Veeco
AFM Probes	VistaProbes	T300R-10