Purification of histag protein using HISTRAP HP (GE). I am working with insect cells (Tn5B) and my antigens are expressed in the nucleus. I am having difficulty lysing the cells because we have no sonicator.
My antigens have already been detected using anti-histine tag antibody. Now, I need to lysis the cells to obtain the antigens and purify them. Physical method like freezing in liquid nitrogen or ultrasonic bath did not work. My antigens stayed in the pellet after 5000 rpm for 30 min (4ºC).
which lysis buffer would be suitable for the column?
For purification, I prepared the following buffers:
Binding Buffer:
500mM NaCl
20mM NaH2PO4
20mM Imidazole
pH 8.0
Elution buffer
500mM NaCl
20mM NaH2PO4
500mM Imidazole
pH 8.0
I still haven't been able to purify anything. What suggestions could you give me?
Its possible you will need denaturing conditions to create soluble proteins. There are a variety of solubilizing conditions you can explore from mild (non-detergent sulfobetaines) to harsh (surfactants or chaotropic agents). Then its just a matter of finding refolding conditions. Are they fancy proteins ? Multiple trans membrane helices or many S-S bond complicate matters.
Do insect cells have chitin in their cell wall? If yes, treat them with chitinases.
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