I used Agilent Calmodulin resin to affinity purify my recombinant protein from Sf9 cell lysate. Following the generic calmodulin resin protocol I found online (no protocol was offered by Agilent), I used 2mM EGTA to elute protein from the resin, but found that all my protein was still bound (after 4 rounds of elution with 1x resin bed volume). Has anyone else had this experience or can suggest alternate approaches?
/Anna
You can try once using a higher concentration of EGTA : Tris buffer pH 7.4, NaCl 50-200 mM, EGTA 5 mM , and maybe use more than just 1 volume of resin bed (ex.3-5)
Calmodulin elution buffer 10X stock buffer (2 M Tris-HCl (pH 7.9), 1 M NaHCO3, 0.2 M EGTA, 7 μL β-mercaptoethanol in 10 mL buffer).
Please look at the following below links which may help you in your analysis:
https://www.chem-agilent.com/pdf/strata/204300.pdf
http://146.189.216.207/contentassets/1fb3bc6b22e943628526421007c75e0f/tap-purification.pdf
Thanks
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