The overall goal of the following experiment is to rapidly obtain amyloid fis from any protein in vitro. This is achieved by dissolving the protein of choice in MES buffer to induce structure misfolding. The protein can then be incubated at high temperature, which promotes the formation of amyloid in the absence of other co-factors.
Alternatively, the protein is cross-linked by EDC in order to generate stabilized soluble protein ligaments, the precursor of amyloid. Finally, non-protein co-factors are mixed with stabilized soluble protein oligomers to allow the formation of hybrid amyloid results are obtained that show native proteins can be readily converted into amyloid confirmation when facilitated by favorable conditions. This method can help answer key questions in protein misfolding diseases, such as how precisely amyloid forms from soluble protein oligomers, and whether different types of amyloid have distinct biological and pathological functions.
To begin, prepare MES buffer as described in the text protocol for both protein only amyloid and stabilized soluble protein. All ligaments then weigh the protein of choice and reconstitute it to 10 milligrams per milliliter in MES buffer. In this demonstration, human serum albumin or HSA is used, incubate the solution at 65 degrees Celsius in a water bath for four hours.
By the end of the period, a white precipitate is visible in the solution. Neutralize the sample with 10%volume per volume. T tris, HCL dialyze the protein in a dialysis cassette overnight to exchange to the desired buffer.
For further analysis, prepare a control sample of native protein by following these steps without heat precipitation. To prepare stabilized soluble protein, all ligaments weigh the protein of choice and reconstitute it to 10 milligrams per milliliter. In MES buffer, leave the protein solution at room temperature for 15 minutes.
Prepare a fresh stock of 20 milligrams per milliliter EDC in MES buffer. Mix the protein and EDC solutions at a ratio of two to five, then incubate at room temperature for two hours following incubation. Neutralize the sample with 10%volume per volume of tris HCL dialyze the protein in a dialysis cassette overnight for exchange to the desired buffer.
For further analysis. Store the stabilized soluble protein all ligament at four degrees Celsius for up to four weeks. This suspension will be used to prepare hybrid amyloids for preparation of both the DNA containing amyloid and the RNA containing amyloid.
Mix, the nucleic acid and stabilized soluble protein all ligament at a one one-to-one ratio. Leave the mixture at four degrees Celsius for at least two hours during this period. Insoluble precipitate is visible in solution when the initial protein concentration is 0.5 milligrams per milliliter or higher.
To prepare the heparin containing amyloid mix heparin and stabilized soluble protein, all ligament at a one-to-one ratio before incubating it at four degrees Celsius for at least two hours. Similar to the nucleic acid containing amyloids in soluble precipitate is visible in solution. When the initial protein concentration is 0.5 milligrams per milliliter or higher, the ability to convert native proteins directly to amyloid relies on the confirmational change incurred at high temperature.
In the MES buffer, precipitation in the solution is a good indication of protein aggregation and possible amyloid formation to allow soluble protein oligomers to stabilize. EDC is used to cross-link the proteins to solidify the oligomeric confirmation induced in MES buffer. As a result, the stabilized protein allgas are multimeric proteins distinct from monomeric native protein or heat induced aggregates.
When separated by SDS page stabilized soluble protein, all ligaments can readily bind to nucleic acids, including both DNA and RNA in a sequence independence manner. Therefore, a gel shift assay can be performed to visualize the direct interaction between the stabilized soluble protein, all ligaments and DNA, which would result in the retarded migration of DNA on an AROS gel. Cytotoxicity is an archetypical character of soluble protein.
All ligaments stabilized soluble protein, all ligaments dose dependently induced cell death that is detectable by fluorescence activated cell sorting. As shown here, RP I 8 2 2 6 cells are extremely susceptible to soluble protein, all ligament induced death that is measured by propidium iodide staining. After watching this video, you should have a good understanding of how to prepare different types of amyloid from any protein of your choice in vitro.
