Visualisatie van Recombinant DNA en eiwit complexen met behulp van Atomic Force Microscopy

The overall goal of this procedure is to image biomolecules such as DNA and proteins in aqueous buffers in real time using atomic force microscopy. This is accomplished by first preparing biomolecules to be imaged. Next, the atomic force microscope is set up and the a FM cantilever probe is positioned.

The sample surface and biomolecules of interest are then imaged and analyzed. Ultimately, results can be obtained that show general size, amount and organization of DNA and proteins and heterogeneous biomolecular complexes in buffered conditions through atomic force microscopy. This method can help answer key questions in the molecular chaperone field, such as what is the STO geometry of chaperones and cos chaperones complex to a client.

Protein Visual demonstration of this method is beneficial as the A FM and Kent lever probe preparation steps may be difficult to learn from printed instructions largely because of the intricacies associated with their physical manipulation. Demonstrating the procedure will be Morgan Shannon and John Gertz, two students from my laboratory To begin isolate the protein or DNA molecules to be imaged and suspend them in an aqueous buffer. After confirming the purity of the sample, incubate it in an a FM absorption buffer on ice to promote adherence of the sample to mica.

For protein samples, use a 10 millimolar heaps buffer. And for DNAA magnesium containing buffer composed of 10 millimolar tris, pH 7.5 10 millimolar sodium chloride, two millimolar magnesium chloride is suitable to mount the A FM probe. Place the liquid cell probe holder onto the corresponding cantilever installation docking station.

Locate the long thick cantilever of the sharp nitride lever probe to be used for imaging. Carefully transfer it to the liquid cell probe holder, ensuring that the tip remains upright. Next, examine the cantilever under a light microscope to ensure it is intact and properly seated in the liquid cell probe holder.

Then carefully remove the a FM head from the instrument dovetail assembly by tightening the neural head clamp screw. Invert the a FM head and firmly push the liquid cell probe holder onto the four pins at the base of the a FM head. Finally, carefully return the a FM head into the instrument of tail assembly.

To further prepare the a FM before imaging samples, move the knobs of the onboard light microscope to locate the cantilever tip, adjust the up and down arrows of the optics controller on the software to bring the cantilever tip into focus. Use the laser adjustment knobs to align the laser to the cantilever tip. This is one of the most technically challenging steps in the protocol Positioning is done manually and requires precise knob adjustment.

Careful monitoring of the laser in the reflection spot and the illumination spot increase the likelihood of successful alignment. Next, adjust the photo detector knobs on the a FM head To center the photo detector. Place a freshly cleaved sheet of MICA attached to a metal a FM specimen disc on the magnetized sample holder atop the A FM holder plate.

Rotate the a FM holder plate so the specimen disc is positioned for the initial imaging. Use the focus surface control of the instrument software to move the a FM head down toward the surface of the MICA specimen disc. Until distinct features on the MICA surface or the tip reflection are in focus, select the tune icon from the instrument software and tune the cantilever.

The resonance frequency of recommended MSNL or SNL probes is 20 to 60 kilohertz. Select a 5%peak offset to image the MICA sample. First, engage the probe onto the MICA surface set initial scan size to 10 micrometers and the scan rate to one hertz.

Capture complete scan images of the 10 micrometer field. Then decrease the scan size to five one and 0.5 micrometers and capture complete images of each field. Disengage the probe by clicking once on the disengage icon on the instrument software.

To image biomolecules of interest, mix five microliters of the prepared sample with 45 microliters of fresh absorption buffer. Carefully add 50 microliters of the 0.5 microgram per milliliter biomolecule containing solution directly onto the MICUs surface. Pause for five minutes to allow the biomolecules in the sample to adhere to the Micah surface.

Then pipette an additional 50 to 100 microliters of absorption buffer into the liquid cell probe holder. Being careful to not touch the probe, a FM head or MICA with the pipette tip, readjust the photo detector and realign the laser to the cantilever as necessary. Then use the instrument software to reengage the probe.

Increase the scan size to identify regions of interest. Once imaging is complete, select the withdraw icon on the instrument software several times to disengage the probe. Finally, use distilled water to thoroughly rinse the liquid cell probe holder and specimen disc.

Then try it with compressed air. An example of an A FM image is shown here. The mica substrate provides a molecularly flat surface onto which DNA and protein can absorb.

Imaging MICA prior to biomolecule samples provides a negative control and an assessment of imaging noise. It also provides a level of assurance that the cantilever is properly tuned and subsequent sample imaging will be successful. Double stranded plasma DNA presumptively super coiled is readily identified by its asymmetric appearance and uniform depositing on the previously unremarkable Micah substrate.

Protein complexes of discrete particle sizes are also uniquely distinguishable from the Micah substrate. Particle size differences indicate sample heterogeneity and can be useful for approximating protein, complex stoic geometry or biochemical activity. The general diagonal shape and consistent orientation of the protein particles observed.

Here is an imaging artifact as proteins would expectedly be oriented on the Micah substrate in a random fashion. Possible causes of the observed artifact or a physical tip abnormality or a FM imaging at two rapid of a scan rate while tip convolution prevents absolute length measurement calculations in the x and Y axis height measurement or Z axis and relative X and Y measurements may be nonetheless useful for estimating biophysical properties of the imaged biomolecules. While attempting this procedure, it's important to remember to accurately tune the cantilever and align the photo detector Following this basic a FM procedure.

Other steps, including using a buffer exchange fluid cell, can be added in order to answer additional questions such as the ability for molecular chaperones to affect the release of steroid receptors from their DNA response elements. After watching this video, you should have a better understanding of how to image DNA proteins and biomolecular complexes using atomic force microscopy in buffered conditions.