2 replies to this topic

[wizzkid]

Hi everyone,

I need an answer to my query. I'm three weeks into the start of my PhD and I want to co-IP proteins of a particular complex.
My supervisor whats me to follow the lab protocol exactly (everything done in the lab needs to be standard). However other research groups are using 150mM NaCl in their lysis buffers. The protocol in my lab is 50 mM NaCl. I know my supervisors point is correct that a lower salt concentration is better for Co-IPing but I cant manage to co-IP the proteins following the lab protocol.
Does salt concentration make any diff

Thanks guys! would really appreciate an answer

[bob1]

150 mM NaCl is approximately 0.9% or "normal saline" which is about the same osmolarity as normal body fluids.  This is more or less ideal for preserving protein structure, if it is dependent on the salt concentration (which it may not be).  However, 50 mM can be used to lyse the cells by osmotic shock, so you might get more of the protein in solution than you would using 150 mM.

I have seen IP protocols with NaCl conc ranging from 0-300 mM.  All of them work to some extent, so I would give it a go with the lab protocol first, and if that doesn't work, then you can think about changing things.

[Ketil.Pedersen]

Co-IP mimics the intracellular conditions for interactions between binding partners. This often requires a lot of optimalization as interactions are often weak, transient (short) as a response to e.g. an activation of a receptor. Each protein complex is different and requires optimized conditions and its hard to predict the conditions for a certain protein-protein complex. Some protein protein interaction requires the presence of DTT and/or MgCl2 to remain intact while this will disrupt other complexes. Increased presence of detergent and salt will generally increase the stringency so fine tuning the stringency is required to provide optimal binding conditions and minimal unspecific binding.(Oeffinger et al., Nature Methods  (2007))

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Ketil Winther Pedersen, Ph.D
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Tel: +47 22 06 11 10 (direct)
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