Author Topic: Co-Immunoprecipitation  (Read 797 times)

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Offline VTimberlake

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Co-Immunoprecipitation
« on: January 08, 2016, 08:55:19 PM »
Functions of Co-Immunoprecipitation(Co-IP): (1)Detection of binding of two target proteins;(2)identification of identify a new binding partner of a particular protein; (3)getting the natural interacting protein complex by separation.

When the cells are lysed under non-denaturing conditions, interaction of protein-protein in intact cells is retained. If use the antibodies of protein x to have Immunoprecipitation with x, protein Y which is bound to protein X also can precipitate. At present, refined protein A is used to bind to beads in argarose previously; after having an antibody reaction with solution containing antigens, protein A in beads can absorb antigens. This technique is usually used to detect binding of two target proteins and identify a new binding partner of a particular protein.

Co-Immunoprecipitation
« on: January 08, 2016, 08:55:19 PM »

Offline KWinslet

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Re: Co-Immunoprecipitation
« Reply #1 on: June 24, 2016, 03:34:38 AM »
If you are looking for the co-ip product. First do the lysis in following buffer (25 mM Tris-HCl pH 7.4 or pH8.0 + 100 mM NaCl+50 mM NaF+ 2mM EDTA+ Protease inhibitor + 0.5% Tx100).  If protein is soluble then Tx100 is very good but in case of  Membrane protein you may have to use 2mM DDM instead to Tx100. Lysis should be done at 4 degree and clear the insoluble fraction by centrifugation  at 2000g/15 min.  Do the co-ip with ab/beads for 1 hr (maximum 90 min). Wash the beads 4x with same lysis buffer and keep the centrifugation speed same.

Elute the protein by adding 50 ul beads (for 1ug antibody per IP) for 95 degree 3 min.

Re: Co-Immunoprecipitation
« Reply #1 on: June 24, 2016, 03:34:38 AM »