Ten μl of a 10-mg ml−1 lipid mixture was dropped onto a circular glass coverslip, air dried and kept under vacuum for at least 1 h. The dried lipid smear was hydrated with 90 μl of GTPase buffer for 30 min, followed by rinsing to peel off the membrane regions that did not adhere to the surface. Two μl of the protein mix (AP180+clathrin) was incubated with the adhered membrane sheet for 30 min, followed by rinsing with GTPase buffer to wash the unbound protein. The protein-binding membrane sheet was then fixed with 4% glutaraldehyde and 1.8% osmium tetroxide for 1 h, followed by washes with GTPase buffer. The sample was then dehydrated by series of ethanol washes (30%, 50%, 70% and 90% ethanol for 5 min each) and further kept overnight under absolute ethanol. The dried sample was then imaged using the Multimode V AFM (Veeco) in tapping mode. Pointprobe Plus tips (Nanosensors, Neuchatel, Switzerland) with non-contact high frequency (C=42 N.m−1, fo=330 kHz) were used for scanning the image.
Pointprobeplus tips
Manufactured by Nanosensors
Sourced in Spain
PointProbePlus tips are designed for use with scanning probe microscopy equipment. They feature a sharp, precise tip for accurate sample interaction and measurement. The tips are made of durable materials to provide reliable performance.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using pointprobeplus tips
1
Lipid Membrane Visualization by AFM
2
Protein Adsorption on Mica for AFM
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Twenty µL a of protein solution at 0.85-6.8 ng/μL were incubated for 4-5 min onto freshly cleaved mica as previously described (1) . The surface was thoroughly rinsed with 6-7 mL of deionized water to remove buffer components.
Finally, the sample was dried under nitrogen air flow.
Samples were scanned under an AFM from Nanotec (Nanotec Electrónica, Madrid, Spain) using PointProbePlus tips, type PPP-NCH (Nanosensors, Neuchâtel, Switzerland). Amplitude Modulation AFM (AM-AFM) mode was used for imaging in air, at room temperature and low humidity. Image processing (general plane subtraction, line flattening) and height profiles were done with WSxM freeware (11) .
Finally, the sample was dried under nitrogen air flow.
Samples were scanned under an AFM from Nanotec (Nanotec Electrónica, Madrid, Spain) using PointProbePlus tips, type PPP-NCH (Nanosensors, Neuchâtel, Switzerland). Amplitude Modulation AFM (AM-AFM) mode was used for imaging in air, at room temperature and low humidity. Image processing (general plane subtraction, line flattening) and height profiles were done with WSxM freeware (11) .
3
Protein Adsorption on Mica via AFM
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20 L of protein solution at 0,85 ng/L were incubated for 4-5 minutes onto freshly cleaved mica. The surface was thoroughly rinsed with 6-7 mL of deionized water to remove buffer components. Finally, the sample was dried under nitrogen air flow [21] .
Samples at acidic and basic pH were prepared in the same way, but pH was previously changed by dilution up to the final protein concentration (0,85 ng/L), with citrate buffer (200 mM NaCl, 20 mM citrate, 1 mM EDTA) when shifting to acid pH or Tris buffer (200 mM NaCl, 20 mM Tris pH 8.5, 1 mM EDTA) for basic pH.
Protein was incubated at 4 ºC during a minimum of 30 minutes before use.
Samples were scanned under an AFM from Nanotec (Nanotec Electrónica, Madrid, Spain) using PointProbePlus tips, type PPP-NCH (Nanosensors, Neuchâtel, Switzerland). Amplitude Modulation AFM (AM-AFM) mode was used for imaging in air, at room temperature and low humidity. Image processing (general plane subtraction, line flattening) and height profiles were done with WSxM freeware [22] .
Samples at acidic and basic pH were prepared in the same way, but pH was previously changed by dilution up to the final protein concentration (0,85 ng/L), with citrate buffer (200 mM NaCl, 20 mM citrate, 1 mM EDTA) when shifting to acid pH or Tris buffer (200 mM NaCl, 20 mM Tris pH 8.5, 1 mM EDTA) for basic pH.
Protein was incubated at 4 ºC during a minimum of 30 minutes before use.
Samples were scanned under an AFM from Nanotec (Nanotec Electrónica, Madrid, Spain) using PointProbePlus tips, type PPP-NCH (Nanosensors, Neuchâtel, Switzerland). Amplitude Modulation AFM (AM-AFM) mode was used for imaging in air, at room temperature and low humidity. Image processing (general plane subtraction, line flattening) and height profiles were done with WSxM freeware [22] .
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