Hello. I am currently working on a project which involves silk fibroin. The problem is , I'm facing gelation in different steps almost each time. I made 4 attempts till today. At the first attempt I was able to obtain %8 silk fibroin but on other 3 attempts I faced gelation problems twice at dialysis stage and once at autoclave. I'm sharing the protocol which I use. I hope you can give me some advice on the issue.
1. Prepare 5 grams of cocoon shell on a petri dish. (Cut them into small pieces)
2. Add 4.24 mg NaCO3 in 2 liters distilled water, dissolve the NaCO3 and heat the solution to 90 celcius degrees.
3. Add the cocoons to the hot distilled water and wait 30 minutes while stirring.
4. Take out the silk and put into a new beaker inside 2 liters of distilled water (at room temperature) for washing.
5. Wash at least 3 times for 20 minutes. (I'm washing it 4 to 6 times and I rinse the silk with distilled water with squeezing it between water changes) (I make sure that the soapy feeling is lost)
6. Put the silk into a petri dish.
7. Let the silk dry at 37 C for 24/36 hours.
8. Take the silk out and weigh it. Cut the dry silk into pieces.
9. Prepare lithium bromide (9.3M) solution and put 4x of the silk's weight:
-The amount of LiBr: [(86.65x9.3)/1000]x4x(Silk's weight)
-The amount of distilled water: 4x(Silk's weight)
10. Dissolve LiBr in distilled water with magnetic stirrer and add it onto the silk in a 50mL beaker.
11. Put the solution into 60 C incubator for 4 hours. (I waited 4 hours for first 3 attempts, then I waited 6 hours at my final attempt which solidified while autoclaving)
12. Put the liquid solution into dialysis sacks ( Sigma-Aldrich D6191-25EA; 12,000 Da MWCO) and tie the sack from up and down. Leave a little space on top.
13. Hang the dialysis sack in 2 liters of distilled water and open the stirrer.
14. Change the water in regular intervals (after 1h, 3h, 6h, 10, 20h) and rinse the LiBr for 2 days.
15. Open the sack and put silk fibroin into a glass bottle.
16. Autoclave.
17. Take 1 mL of autoclaved silk fibroin and put into a centrifuge tube without closing the cap. (Note the weight of centrifuge tube and the silk fibroin)
18. Put the tube into the incubator (60 C degrees) overnight.
19. Weigh the tube. Calculate the amount of dry matter and then calculate the percentage of silk fibroin.
Details:
-The lab's temperature is around 26 C degrees.
- I use different stirrers to avoid heating the water at step 5.
-The liquid I obtain after LiBr application is very viscous compared to the videos I watched.
-The cocoons are present at the lab for 4 years and they are being kept in room temperature in a drawer inside a plastic bag.
First thing during dialysis, gelation is a problem. I also faced many times but now what u have to do....increase the time of degumming process (45min). One thing if you required silk fibroin solution then there is no need of autoclave, it may harm the silk property. During the dialysis steering is not required. Just keep your water container in which the silk fibroin sacks are hanged in incubator at 35 to 40 C temperature. I am quite sure that it will not be gelled again. just try it and tell me the result.
Hi Tayfun
Autoclave step is not required here. I hope you are following the same process:
Boil cut cocoons in 0.02 M Na2CO3 for 60 min and washed them with deionized water to remove sericin from the silk fibers, and then dry the degummed fibers. These degummed fibers will be dissolved in 9.3M LiBr at 60 degree for 4 h. The regenerated silk fibroin solution will then dialyzed in cellulose tube (12 kDa, MWCO) against deionized water for 48 h to remove the remaining lithium bromide. Fibroin solution will be collected after centrifugation.
Thanks
Unfortunately I have to autoclave silk fibroin. I use it on stem cell culture so it needs to be sterile. I acquired new batch of LiBr from a colleague since mine was really old and I also prolongued the degumming to 45 mins and performed the dialysis as Mr. Singh adviced and it worked really well. The silk fibroin also survived from autoclave. Thank you.
Dear Tayfun Dikmen,
Generally, Gelation involves the formation of self-similar aggregates with a growth rate that increases exponentially. Apply initially couple of constraint conditions. Some of points are advised enlisted here.
(1)Freeze-drying- this will give gel a foam with a porosity of 80.90%, a water uptake capacity of 90% and a swelling index of 8.5-10. Gelation time and the properties of hydrogels and porous foams could be controlled by the ratios of RSF and non-solvent concentration as well as by the type of non-solvent and incubation temperature.
(2) PH must be between 2-4. If less than 2 it will defiantly form gelatin as pH is negative concentration of hydrogen algorithm. RSF (regenerated silk fibroin) hydrogels and porous foams can possibly be used for the encapsulation of cells and/or for the controlled release of both hydrophilic and hydrophobic drugs.
(3) Concentration must between ranges 0.5-10gL-1. Nonsolvent concentration can control the porous foam.
(4) Temperature between 5-70 0 C is advisable.
(5) Turbidity, rheology, light scattering and circular dichroism are other issues. Custody minimum turbidity will avoid early gelation.
Ashish
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