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Washing Steps in Immunoprecipitation


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#1 Mighty Mouse

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Posted 13 July 2010 - 06:32 PM

Just curious if someone can explain to me the logic behind using various bead washing steps during immunoprecipitation protocols...I understand the purpose, to reduce non-specific binding to the antibody, but how?

Typically what I've seen is washes with the following:

Low Salt (150 mM NaCl + tris, EDTA, tritonX and SDS)
High Salt (500 mM NaCl + "")
LiCl (250 mM LiCl, deoxycholate, EDTA, tris, and NP40)
TE

Just curious as to how these various washes assist in removing non-specific binding.

Thanks

MM
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#2 Mighty Mouse

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Posted 17 August 2010 - 06:37 PM

Just curious if someone can explain to me the logic behind using various bead washing steps during immunoprecipitation protocols...I understand the purpose, to reduce non-specific binding to the antibody, but how?

Typically what I've seen is washes with the following:

Low Salt (150 mM NaCl + tris, EDTA, tritonX and SDS)
High Salt (500 mM NaCl + "")
LiCl (250 mM LiCl, deoxycholate, EDTA, tris, and NP40)
TE

Just curious as to how these various washes assist in removing non-specific binding.

Thanks

MM


Any thoughts anyone?
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#3 Felipillo

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Posted 17 August 2010 - 10:15 PM

You are from low stringent conditions to high, becouse in mild stringent conditions the polar interactions could be very inespecific, cause the water can supply hydrogen bonds in different sites for two molecules, so if you add salts, the water are sorrounding the ions, not your interacting proteins.

Edited by Felipillo, 17 August 2010 - 10:16 PM.

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