The overall goal of this procedure is to generate peptide based assemblies in the form of biomolecular necklaces. This is accomplished by first preparing stock solutions by dissolving the Diphenyl alanine and its Bach protected analog separately in the appropriate amount of HFP to a final concentration of 100 milligrams per milliliter. The second step is to blend the peptide stock solutions in a five to three ratio by mixing 10 microliters of the Diphenyl alanine peptide solution with six microliters of the Bach protected Diphenyl alanine peptide solution.
Next, the blended peptide stock solution is added to freshly prepared 50%ethanol to give a final concentration of five milligrams per milliliter of diphenyl alanine and three milligrams per milliliter for Bach protected diphenyl alanine respectively. Effectively ultimately scanning electron microscopy is used to show the biomolecular necklaces co assembled by the two peptides. The main advantage of using processes of self-assembly like the one demonstrated here, is that by spontaneous progression, it is possible to generate complex structure.
In vitro here represent the self-assembly of peptide, specifically the co assembly of Dipeptides demonstrating this procedure will be nuran, a graduate student former laboratory. To begin the procedure for the self-assembly of Diphenyl alanine into tubular structures, prepare 100 milligrams per milliliter peptide stock solution by dissolving Diphenyl alanine peptide in HFP. To ensure the homogeneity of the solution, mix it with a vortex mixer for a few seconds and set it aside on the bench for a few minutes until the peptide is completely dissolved and the solution is clear.
Dilute the peptide stock solution to a final concentration of two milligrams per milliliter in triple distilled water. Keep the solution at room temperature for 24 hours. For the co assembly procedure, dissolved two milligrams of diphenyl alanine peptide and one milligram of Bach protected diphenyl alanine peptide separately in HFP to a concentration of 100 milligrams per milliliter.
Then mix the solutions with a vortex mixer for a few seconds and set them aside on the bench for a few minutes until the peptides are completely dissolved and the solutions are clear. Blend the peptide stock solutions in a five to three ratio by mixing 10 microliters of the Diphenyl alanine peptide solution with six microliters of the Bach protected diphenyl alanine peptide solution. Then mix the solution using a vortex mixer.
Following this, add eight microliters of the blended peptide stock solution to 92 microliters of freshly prepared 50%ethanol to give a final concentration of five milligrams per milliliter for diphenyl alanine, and three milligrams per milliliter. For Bach protected diphenyl alanine. Use a pipette to gently mix the solution.
After 24 hours of incubation at room temperature, apply a 10 microliter drop of the peptide solution on a glass cover slip and allow it to dry at room temperature. Coat the sample on the glass cover slip with a thin layer of gold using a sputter coter for 90 seconds. When finished, image the assemblies using SEM operating at 10 to 20 kilovolts.
For TEM analysis, place a 10 microliter drop of peptide solution on a 200 mesh copper grid covered with carbon and stabilized by a polymer film support. After one minute, remove the excess fluid using filter paper. Next, transfer a 2%urinal acetate solution into a syringe and filter it using a zero point 22 micrometer filter unit.
To stay in the sample place a 10 microliter drop of urinal acetate solution on the grid. After 30 seconds, remove excess fluid using filter paper image the sample on the grid by TEM operating at 120 kilovolts. For FTIR analysis, apply a 30 microliter drop of the peptide solution to a calcium fluoride window.
Allow the solution to dry at room temperature in order to suppress the water signal in the FTIR spectrum. Place a drop of deuterium oxide on the dry peptide sample. After drying the sample in a desiccate under vacuum, repeat the previous steps twice to ensure maximal hydrogen to deuterium exchange.
Finally, record the FTIR spectra using a deuterated triglycine sulfate detector. Determine the transmittance minimal values using the software supplied with the instrument to demonstrate the formation of ordered structures by self-assembly of peptides. The co assembly of two simple aromatic peptides were prepared and characterized using SEM and TEM analysis.
The blended peptides formed an architecture of spherical assemblies with a diameter of several microns connected by elongated structures with a diameter of a few hundred nanometers due to the high resemblance in morphology to beat its strings. These structures are termed molecular necklaces. A FM analysis of these structures clearly demonstrates their three-dimensional arrangement.
In addition, SEM analysis of different regions of various samples indicates that this process occurred with high yield. FTIR analysis provides information on the secondary structure of the peptides assemblies. The absorbent spectrum of the amide.
One band of the spherical assemblies formed by the Bach protected diphenyl alanine peptide in ethanol showed a single amide peak at 1, 657 reciprocal centimeters indicating an alpha helix confirmation. The tubular structures formed by the diphenyl alanine peptide in ethanol showed two distinctive peaks at 1, 613 reciprocal centimeters and 1, 682 reciprocal centimeters, which correlate with a beta sheet secondary structure. The FTIR spectrum of the biomolecular necklaces formed by the co assembly of the two peptides deferred from the peak assignment for each individual peptide.
The first peak at 1, 653 reciprocal centimeters corresponds with an alpha helix structure and the second peak at 1, 684 reciprocal centimeters relates to a beta turn confirmation. The difference between the various spectra indicates a unique structure for biomolecular necklaces. After watching this video, you should have a good understanding on how to generate peptide based assembly by dye peptide.
This method can be applied to other biomolecule easily in order to form complex structure. Don't forget that walking with organic solvents can be extremely hazardous.Gloves. He lab coat and safety glasses should always be worn while performing this procedure.
