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Periplasmic Prep. failure - Help needed (Jun/29/2011 )

Hi all,

Im struggling at the moment to extract recombinant protein from the periplam of E-coli BL21. Only small amounts can be extracted and when a standard sandwhich ELISA is performed on them the quantities vary from none existant to way above the background. To determine if the protein is being expressed at all I burst open remaining pellet after each periprep and tested the contents. This seemed to show high levels of binding during the ELISA. I find this higly unusual as the protein of interest has lots of disulphide bonds which to my knowledge require insertion into the periplasmic space for correct folding.


Brief method
Day 1 O/N growth single colonies 1ml 2TY Amp Glu
Day 2 Hedgehog new media, grow OD600 of 0.5 induce with Arabinose and incubate O/N 22C at 280rpm
Day 3 Periplasmic extraction,spin down O/N culture and add 200ul: 20% sucrose 30mM Tris-HCL 0.1mM EDTA. leave 10 mins, spin down and add 100ul MgSO4, leave 10 mins, spin and remove supernatant for use in standard HRP ELISA

Any help to make this successful and consistent would be greatly appreciated. As you can imagine so much time has now been wasted in performing these periplasmic preperations.

Thanks

-ChrisResearcher-

ChrisResearcher on Wed Jun 29 15:25:48 2011 said:


Hi all,

Im struggling at the moment to extract recombinant protein from the periplam of E-coli BL21. Only small amounts can be extracted and when a standard sandwhich ELISA is performed on them the quantities vary from none existant to way above the background. To determine if the protein is being expressed at all I burst open remaining pellet after each periprep and tested the contents. This seemed to show high levels of binding during the ELISA. I find this higly unusual as the protein of interest has lots of disulphide bonds which to my knowledge require insertion into the periplasmic space for correct folding.


Brief method
Day 1 O/N growth single colonies 1ml 2TY Amp Glu
Day 2 Hedgehog new media, grow OD600 of 0.5 induce with Arabinose and incubate O/N 22C at 280rpm
Day 3 Periplasmic extraction,spin down O/N culture and add 200ul: 20% sucrose 30mM Tris-HCL 0.1mM EDTA. leave 10 mins, spin down and add 100ul MgSO4, leave 10 mins, spin and remove supernatant for use in standard HRP ELISA

Any help to make this successful and consistent would be greatly appreciated. As you can imagine so much time has now been wasted in performing these periplasmic preperations.

Thanks

Hola, I suposse that when you put the bacteria in sucrose and Mg solns. you resuspend the pellet. when I made osmotic shock the swolen step was a bit long as 20-30 min and the Mg solution was 1-5 mM. Buena suerte

-protolder-