I want to purify the protein from E.coli. My proetin of interest is expressed in pellet .
so i used lysis buffer( 50mM tris, 6M GnHCL, 20 mM imidazole and 500 mM nacl),.
After stripping , clean up and recharging . My protein lossely bind to coloumn , in the wash fraction i found most of my protein , not able to purify it. any suggestions?
Dear Seema
some question:
how you performed stripping, clean and recharging of the coloumn?
Is ir not work for a new protein oreparation ir to bind the flow though (unbunded protein) from the previous preparation?
best regards
manuele
Jorgaq Pata i used the same lysis buffer before it , for the same protein but a different batch.
Manuele Martinelli Sir, i stripped the column with 5CV of buffer having 20mM sodium dibasic phosphate ,0.5M NaCl and 50mM EDTA ph=7.4,
then wash with 5 cv of lysis buffer and then 5cv of ddH2O.
For clean up - after that again wash with 5cv of 1.5M NaCl ,5cv of lysis buffer,then 10cv of ddH2O
passes 5cv of 1M NaOH , wash with 10cv of ddH2O.
then pass 5cv of 30% isopropanol and wash with 10 cv of ddH2O and store it in 20% ethanol
please help
.
Manuele Martinelli Sir , its not binding for the same protein from new batch , yes came out in flow through (unbounded protein).
Dear Seema
thwo point:
1) after washing with Naoh and isopropanol did you readed the metal? because Noah will strip the metal and if you would like that the coloumn work again metal have to be present.
2) i think that your stripping, cleaning regeneratuin coloumn is too strong, in imac edta cleaning is enough.
you can find a detailed protocol that i routunelly uding on my blog ProteoCool ( https://proteocool.blogspot.com/)
ProteoCool n°25
(A simple way to clean and regenerate the IMAC coloumns) available at Page7. Try it and let me know if you solve your problems.
good luck
Manuele
Manuele Martinelli sir after cleaning with isoproanol , i recharge them with 0.1 M NiSO4. Thanks for advice i will try it and let you know.
Manuele Martinelli sir i tried you protocol , but after regeneration of the column, all protein bind to it , there is no specific band are for my protein.
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