1. What is "cutting"?
Cutting is a step in the production of peptides. Chemically, it is the separation of peptides from the resin, and removal of the excess temporary protective groups from the polypeptide side chain and the main chain.
2.The front and rear process of cutting
3. Prepare the resin for cutting
All it takes is the solvent in the resin to be washed off and fully dry. Its ideal state is that: it is like dry sand, and it is not easy to stick to the surfaces such as bottles or paper.
4. How to choose and configure the cutting fluid
The information is also available on the Internet, choosing different types of cutting fluid according to different polypeptide sequences.
Here are some common ratios of the cutting fluid.
1) TFA: H2O=95% : 5%, TFA: H2O=97.5% : 2.5%
2) TFA: H2O: TIS=95% : 2.5% : 2.5%
3) trifluoroacetic acid: fennel sulfide: 1, 2-ethylenediol: phenol: water = 87.5:5:2. 5:2. 5
The first two are suitable for non-oxidizable amino acids, such as Trp, Cys, and Met. The third one can be used in both situations. (1 g resin with 8 ml of cutting fluid)
And of course, there are a lot of special cutting methods that will improve the yield, for specific questions, please contact us at 187681771717 ( the WeChat is the same with the phone number ).
5. Cutting process
The regular time is about 2.5 hours, and it's going to keep oscillating, the temperature is room temperature. Some of the polypeptides that are easy to oxidize or deteriorate, we also need to exclude the air in the container (replacing it with nitrogen), sometimes need to properly reduce the temperature of environment, such as 0 to 10 ℃.
6. Filter and collect filtrate
There are many ways of filtering. Each lab has its own method. The main part is that the filtrate filter liquor contains the polypeptide and some side chain protection groups that are cut down. Some of the protective groups that are not soluble in TFA, such as Pbf, they are white floating objects that are filtered out with the resin.
7. Precipitation, centrifugation, washing
I think that this step is the one that most affects yields, if we don't handle it well, a lot of peptides will be lost.
1) solvent: the solvent used for precipitation is ice ether, and the ice here means that the temperature of ether should be very low, for example, in the refrigerator of -20 degrees Celsius. Of course, if you use a normal temperature diethyl ether, that's fine. The volume ratio of ether and filtrate is 6. 1.
2) instrument: a centrifuge capable of 3000 revolutions per minute.
3) method: in the case of time permitting, I recommend that the filtrate be added slowly to 6 times of the volume of ice ether, and it can be used to stir ice ether with a magnetic mixer. The goal is to get the filtered filtrate immediately out of the polypeptide.
4) centrifugal: centrifuge the solution that has already been precipitated out and turned white.
5) wash: we generally recommended washing 3 times, and each time the solution should be stirred evenly.
8. Drain and get the crude peptide
After three times of centrifugal peptide, all kinds of side chain protection groups are basically washed by ether. Just put the wet polypeptide in the vacuum and drain. (don't put in the incubator)