3 replies to this topic

[timeracer]

Hi, everybody.
I am overexpressing  a protein ~90kD using pET15b, BL21(plysS), easily soluble. But the lysate is quite viscid after sonication, not easy to clarify. After 2 consecutive centrifugation at 15000g 30min, the sample is still hard to go through  a 0.45  filter.

So i am planning to use DNase I to eliminate the nucleic acids in the crude extract. But DNase I might not work in start buffer (10mM phosphate, 500mM NaCl, 10 mM imidazole,etc.) Can I just use  20mM Tris to sonicate the cells and add Mg2+, DNase I ,then load directly onto Histrap column which is equlibriumed by start buffer?

I know that generally, the composition of the sample buffer should be the same or similar to that of the start buffer, so I've no idea whether this would be OK. Could anybody give a clue?

[T C]

Hey,

Option 1: dilute with buffer...no harm

Option 2: Use 0.1 % polyethylene imine to precipitate DNA....works in all buffers.

Best,
TC

timeracer, on Jul 20 2009, 08:29 PM, said:

Hi, everybody.
I am overexpressing  a protein ~90kD using pET15b, BL21(plysS), easily soluble. But the lysate is quite viscid after sonication, not easy to clarify. After 2 consecutive centrifugation at 15000g 30min, the sample is still hard to go through  a 0.45  filter.

So i am planning to use DNase I to eliminate the nucleic acids in the crude extract. But DNase I might not work in start buffer (10mM phosphate, 500mM NaCl, 10 mM imidazole,etc.) Can I just use  20mM Tris to sonicate the cells and add Mg2+, DNase I ,then load directly onto Histrap column which is equlibriumed by start buffer?

I know that generally, the composition of the sample buffer should be the same or similar to that of the start buffer, so I've no idea whether this would be OK. Could anybody give a clue?

[timeracer]

T C, on Jul 21 2009, 12:58 AM, said:

Hey,

Option 1: dilute with buffer...no harm

Option 2: Use 0.1 % polyethylene imine to precipitate DNA....works in all buffers.

Best,
TC

Thanks, but my protein is likely to bind nucleic acids, so I'm afraid option 2 would not work because my protein might precipitate with PEI-DNA/RNA complex.

[Luria Bertani]

Hmm. You might be able to use French Press for that, though they still do reccomend DNAse.

EMBL Protein Purification Core Facility has some tips on what you can add.
http://www.pepcore.e...na_removal.html

Alternatively, you may be better performing your expression in a cell-free system? Get some advice first...