I'm working with a mRNA-ribosome-nascent polypeptide complex and I want to perform immunoprecipitation to purify the complex of interest (my nascent polypeptide bears an N-terminal tag).
First, I purify ribosomal fraction from total lysate. Usually a stringent concentration condition, 500 mM KCl buffer, is used in order to get the ribosome core. Because I'm seeking for ribosome associated factors such as proteins and translated mRNA as well, I use a much less stringent condition, that is 25 mM KCl.
I've been looking around and it seems that all buffers used in IP experiments contain at least 100-150 NaCl (or KCl).
I'd like to perform IP with a 25 mM concentration in order not to lose ribosome associated factors, does anyone know if this could prevent the success of the experiment? Have you ever tried to immunoprecipitate at less than 100 mM NaCl (or KCl)? Is that concentration of crucial importance or can one keep it lower?
Thank you so much.
Hi,
People generally use 100-150 mM salt in IP experiments because the physiological salt concentration falls within that range (approximately 137 mM). I think lowering salt conc below 100 mM would only increase the chance of nonspecific interactions, although the optimal condition for your experiments should be determined empirically.
Salt concentration is critical for IPs, and is often something that needs to be optimized on a per-experiment basis. I have successfully immunoprecipitated protein with as little of 50 mM of salt, and as high as 550 mM salt. It really depends on which specific protein you're trying to IP. Generally, increasing the salt concentration makes your IP more stringent and reduces nonspecific binding.
You're best course of action is probably to do a side-by-side experiment. One where you do a low salt (25 mM) concentration, and one with a higher concentration.
Why does salt concentration have an effect on the stringency of the washes?
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