0.1-0.25% Sodium Deoxycholate vs 420mM NaCl in TrionX-100 lysis buffer. Which is - (May/19/2015 )

Dear All,

I want lyse whole cell including nucleus of SY5Y cell without mechanical disrupting for Co-IP.

I have read many resources, and it can be done by either adding 0.1-0.25% Sodium Deoxycholate or increase NaCl to 420mM, in TritonX100 lysis buffer (50 mM Tris•HCl, pH 8.5, 150 mM NaCl, 1%TritonX100).

Which one is better to lyse whole cell for Co-IP?

Thank you

Cheerios,

The answer depends on your protein. You should optimise the conditions to suit. However, in general higher salt concentrations can disrupt protein:protein interactions, as can detergents.... stay as close to physiological conditions as you can.

bob1 on Tue May 19 09:03:04 2015 said:

The answer depends on your protein. You should optimise the conditions to suit. However, in general higher salt concentrations can disrupt protein:protein interactions, as can detergents.... stay as close to physiological conditions as you can.

Oh God. I'd like to lyse nuclear membrane to collect nuclear protein. Non-ionic detergent alone like Triton X-100 won't break the membrane. I know denatuirng detergent can break up some protein-protein interaction. Just know today that high salt can do too.

Do you homuch NaCl would consider it too high? Thank you

IPs are usually done in 300 mM NaCl  or less (or equivalent osmolarity with other ionic solutions), physiological osmolarity is 150 mM NaCl, which equates to 0.9% saline or normal saline. You need to determine which concentrations you can work with for your protein.

bob1 on Tue May 19 20:55:36 2015 said:

IPs are usually done in 300 mM NaCl  or less (or equivalent osmolarity with other ionic solutions), physiological osmolarity is 150 mM NaCl, which equates to 0.9% saline or normal saline. You need to determine which concentrations you can work with for your protein.

Thank you Bob1