Silk Fibroin
The silk fibroin contains a fair number of aspartic (0.5 mol%, ~25 residues) and glutamic acids (0.6 mol%, ~30 residues),as well as a small number of lysine residues (0.2 mol%, ~12 residues).
We use the dry down method. (Weigh out a certain amount of silk fibroin and leave in a 60C oven overnight, and weigh the left-over protein).
The silk we supply is a partially hydrolyzed silk fibroin II solution. Whole cocoons, harvested from domesticated Bombyx mori in China, are degummed and processed into a liquid protein solution using heavy inorganic salts. The solution is then desalted, tested, and packaged. One of the key features of our silk fibroin solution is the preservation of the fibroin light chain at around 25 kDa as shown on the gel.
Prepare 1- 2 Liters of 10% wt./vol. PEG solution and then dialyze the 5% fibroin solution overnight in a 3 kDa dialysis cassette overnight (~12 hrs). This should get you to between 10% - 15% fibroin.
Otherwise, the 'quick and dirty' way to do this is allow the water to evaporate from the Silk Fibroin 5% solution in a clean bench. You can dispense the Silk Fibroin solution into a large weigh dish to increase the evaporative surface and then check the dry weight of the fibroin protein periodically. This method can concentrate the solution significantly by the end of the work day.
The Silk Fibroin Protein that we offer is in the ‘regenerated’ form. This material is derived from the silk worm cocoon and then processed and solubilized into an aqueous form.
The silk is a partially hydrolyzed solution of fibroin heavy and light chain proteins. There will be some free amino acids present due to this hydrolysis, but the large majority of the material consists of the former two elements (heavy and light chains).
The silk solution is made from de-sericinized (or degummed it is sometime called) silk fiber. We remove the sericin via our extraction process.
Yes.
A coating allowed to air dry, or a shorter (15-30 minutes) water annealing.
While the native fibroin proteins exist in a 6:6:1 ratio, this composition is almost certainly not preserved during the manufacturing process that are used to create aqueous fibroin solution. This is because the heavy and light chains of fibroin have considerably different amino acid composition and hydrophobicity, rendering them unequally prone to hydrolysis during sericin degumming. This can be seen on the electrophoretic gel from the solution, where the heavy chain is seen as a smear, but the light chain is a relatively preserved band. We have not endeavored into determining the extent of p25 hydrolysis.
Yes.
It is best to maintain the product at -70 C. Any unused solution should be divided into smaller aliquots and frozen. Try to thaw/refreeze as few times as possible.
The 5% solution has a viscosity of ~12cp. When diluted in water to 2.5%, the viscosity is ~6cp.
Yes, overmixing can alter the physical properties of the solution, the solution is sensitive to mechanical stresses such as shear force. For example, it is possible to induce gelation of the Silk Fibroin Solution by simply vortexing it for 10-15 minutes. Aliquoting the solution or more gentle mixing processes would help reduce changes to the bulk solution.
Lyophilization is not recommended. Unfortunately lyophilization will drive irreversible beta sheet formation under standard conditions.
Fibroin can typically be reconstituted to around 20% protein before it starts to crash out of solution.
If your Silk Fibroin has been stored at a temperature warmer than -20C for too long, it may form aggregates. Unfortunately, due to the tight binding nature of beta-sheets, it is very difficult to redissolve aggregates. We have also tried using LiBr, which was unsuccessful.
No - it is all protein/amino acid.
Yes. The results can be found here.
The silk backbone itself is enzymatically degradable, so some sort of cocktail with high concentration proteinase K should do the work.
