The second purpose of my PhD is to purify and identify the protein partner of protein “A”. I am developing a proteomics approach in 2 steps, 1) purification of cell surface proteins by biotin/avidin affinity, elution in presence of biotin (2mM, overnight) and 2) Immunoprecipitation of protein “A”-associated protein complexes. I perform the proteomic approach using COS-7 cells overexpressing protein “A”. This two-step purification approach is efficient to specifically purify plasma membrane protein “A” and the putative associated protein complexes, I recover the protein “A” at the end of the purification.
HOWEVER, I have a problem when I am performing exactly the same proteomic approach in my model cells, human macrophages. Using the same protocol in macrophages, protein “A” remains bound to avidin beads when I perform elution with 2mM biotin.
I already considered higher biotin concentration (5mM and 10mM) without successful elution of protein “A”.
Had some people already meet this problem? Have you got some ideas?
Please help me, thank you very much.
Charlotte
Yes, but you need monomeric avidin beads. Normal avidin (tetrameric protein) binds biotinylated protein too tight to be elution by biotin. monomeric avidin beads (from Life Biotech, Catalog number: 20228) is specially modified for biotin elution purpose. Hope that helps.
Try with glycin 0.2 M pH 1.5 and then imediately neutralize with unbuffered Tris 1M
Thank you Claude Alban, but glycin is too strong to eluate my protein without breaking its partners because I perform an immunoprecipitation after elution. I need a soft elution.
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