Hello, I am interested in producing purified Cas9 in my lab.
But we are not dealing with proteins so we don't have proper settings for protein purification.
It seems a step using chromatography is necessary for purification.
Is there any protocol for Cas9 purification without using special equipments other than centrifuger? I think it's almost impossible without the chromatography work but...
well, you will need to buy some resin, or disposable columns, have a plasmid encoding for Cas-9 (found in addgene) but it is pretty easy otherwise : check this paper :
https://zenodo.org/record/1325937#.X5ab94hKhPZ
I think that a single step purification may be enough depending on your needs.
You can use His-tag system and this system is not complicated procedure moreover you do not need any special equipments for this experimentation only you should purchased His-Tag kit but first of all you must fused your Cas9 protein with 6 or 8 or 10 Histidine residues but generally 6 histidine residues is enough for the enzyme prufication procedures. For executing fusion procedure first of all you should design primers with 6 His residues but there are some important situations for designing primers. These are;
1) First of all check the active sites of the Cas9 enzyme and the active sites of this enzyme are HNH and RuvC. You can check these sites in uniprot database
2) You should determine where the active sites are close, however the RuvC site is very close to N terminus. When you monitor in uniprot database you will realize this situation so you should add Histidine residues to the C terminus of your Cas9 enzyme. Owing to the fact that If the histidine residues exist far away from the active site then the enzyme activity is protected.
3) To add C terminal Histidine residues you must design your reverse primer without using stop codon of the Cas9 gene sequence
4) Check your primers a special programs such as sigma oligo evaluator
Primer Designing
In reverse primer you ought to add 6 histidine residues and then add a stop codon sequence. I mean that XXXXXXXXXX-HIS-HIS-HIS-HIS-HIS-HIS-STOP CODON (X represents your Cas9 sequence.)
In forward primer you do not need to add anything this primer directly design according to the gene sequence.
After the standart PCR procedure you can acquire a Cas9 with 6 histidine residues at the end of the gene so when this gene is expressed in the cell you can obtain Cas9 protein with 6 Histidine residues at C terminus finally you can isolate your enzyme by using His-Tag kit.
If you want to eliminate these procedures then you can purchase a plasmid vector that contains Cas9 gene with 6 histidine sequences at 3' end of the gene and then you can introduce your plasmid into the BL21(DE3) E.coli serotype to produce your enzyme and then you can isolate your enzyme by using His-tag kit.
I hope I could help you. Good Luck.
If your protein is his tagged then you can get the protein with much purity but for that you need the ni-nta beads.. but before moving in any experiment the motif behind that should be clear.. because if the purified protein is to be used for general purposes then his tag purification is good to go with.. but if it is for any such physico chemico characterization at fine level you have to go for HPLC
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