The overall goal of these procedures is to show how collagen monomer can be used to reproducibly, make three different kinds of higher order collagen structures. Native collagen is made by raising the pH of the solution of collagen monomer, fibrous long spacing. Collagen is made by combining collagen monomer with alpha one acid glycoprotein and segmental long spacing.
Collagen is made by mixing collagen monomer with a TP.Ultimately, atomic force microscopy is used to characterize the three different types of collagen structures that are formed Formed. The main advantage of these protocols over previously published procedures is that they're simpler and very reproducible in forming the desired collagen structures. These protocols have been developed in our lab over the past 10 years, taking advantage of a reliable commercial source of collagen monomer as a starting material demonstrating the procedure will be Calvin Chang, a graduate student from my laboratory, In order to create native collagen fibers with clear 67 nanometer DB banding.
Start by pre warming a heat block to 37 degree Celsius. Next, prepare the buffer solution in a micro centrifuge tube at four microliters of a three milligram per milliliter collagen monomer solution and mix well. The resulting solution should be clear and colorless.
Place the mixture on the 37 degree Celsius prewarm heat block for three to four hours in order to form the native collagen fibrils. The final solution should be slightly cloudy and will contain mainly native collagen fibrils to prepare fibrous long spacing collagen. First place, one milliliter of a three milligram per milliliter collagen monomer solution into a 12 to 14 kilodalton molecular weight cutoff membrane.
Next dialyze against 400 milliliters of water, changing the water four times over a 24 hour period. Next, combine 20 microliters of the dialyzed collagen monomer with 20 microliters of ultra pure water and 20 microliters of three milligrams per milliliter. Alpha one acid glycoprotein in water together into a micro centrifuge tube and mix.
Allow the fibrous lung spacing collagen to assemble at room temperature over a period of 30 minutes. During this time, the solution will go from clear to cloudy to form sick mental long spacing.Collagen. First, prepare the acidic buffer in a micro centrifuge tube.
Add 40 microliters of 10 milligrams per milliliter, a TP in water to the buffer solution and mix. Well finally add 33 microliters of a three milligram per milliliter collagen monomer solution that is in 0.01. Normal hydrochloric acid and mix to combine.
Allow the segmental long spacing collagen to assemble at room temperature over a period of two hours. The final solution will be clear in this preparation, unlike the other two forms of collagen described. To obtain a clean flat surface for atomic force microscopy or a FM, attach a piece of sticky tape to an A FM substrate made of MICA and peel away at least one layer.
Check the tape to confirm the removal of an entire layer. Once a proper surface has been obtained, apply 20 microliters of the collagen FI solution onto the mica substrate and leave for five minutes. After five minutes, rinse away excess fibrils by adding water to the edge of the MICA substrate and letting it flow across the sample for 10 to 15 seconds.
Do not add water directly to the sample area. Then dry the surface using a gentle stream of nitrogen gas from one edge of the substrate. Taking care not to aim the stream at the center of the sample.
Check the sample under an optical microscope at 200 x magnification the native and fibrous long spacing. Collagen samples show clumps of fibro while the segmental long spacing collagen is not visible. The unique characteristics of the different collagen are best observed.
Using an A FM, perform an initial 100 by 100 micrometer square scan and then zoom in to scan sizes of 10 by 10 micrometer square, and finally two by two micrometer square. To observe the banding periodicity of native and fibrous lung spacing. Collagen were the finer features of segmental lung spacing collagen.
For native collagen fis, run the atomic force microscope in intermittent contact mode using a silicon a FM probe for fibrous long spacing and segmental long spacing. Collagen fis run the atomic force microscope in contact mode and use silicon nitrite a FM probes for best results. Check at least two other regions on the collagen sample to verify that the initial scans are representative.
The high spatial resolution of a FM is ideal for characterizing the different preparations of collagen described in this video. It easily distinguishes native collagen fibers, fibrous long spacing collagen, and segmental long spacing collagen optical microscopy techniques such as differential interference contrast microscopy show the fis but are unable to show the nanometer scale features, which differentiate the three types of collagen prepared in these methods. In a typical sample, a random 100 by 100 micrometer square scan by a FM shows at least a few fibrils that are five to 50 microns long individual separated.
Collagen fis can be easily identified at this stage, zooming in to attend by 10 micrometer square scan size shows that all the fis are banded. In order to accurately measure the banding periodicity, it is best to zoom into a two by two micrometer square scan size. Zooming into a 10 by 10 micrometer square scan size shows that all the fibrils are banded.
In order to accurately measure the banding periodicity, it is best to zoom into a two by two micrometer square scan size. The banding period increases when the collagen is prepared according to the long spacing collagen procedure, producing 270 nanometer repeats along the fiber length. In a typical sample, a random 100 by 100 micrometer square scan by a FM shows at least a few fibrils.
The banding periodicity can be seen by zooming into a 10 by 10 micrometer square scan and is easily measurable with a two by two micrometer square scan. Finally, the segmental long spacing collagen does not form fibers at all, but individual segments. In a typical sample, a random 100 by 100 micrometer square scan by a FM shows many individual dots.
A closer 10 by 10 micrometer square scan shows several SLS crystalite and a two by two micrometer square scan shows the finer structure of an SLS crystal light. After watching this video, you should have a good understanding of how to make uniform mature native FLS and SLS collagen constructs from commercially available collagen monomer. Collagen is a biocompatible material that is useful as a substrate or as a scaffold for growing cells and tissues.
This procedure will enable you to start with desirable collagen structures for these and other applications.
