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I am about to use these beads after pull down of the flag protein.
I am washing them in RIPA and PBS and was wondering at which speed you recommend to centrifuge to pellet the beads. Is it fine using 14,000 g for 4 mins, or is it enough 30’’? I have just realised that as I am doing co-ip, 4 mins centrifugation may be too harsh on the protein complexes? I have used in the passed magnetic beads so I didn’t need to centrifuge.
Asked on Nov 09 2012
Thank you for contacting us.
If you are doing co-IP, it is not recommended to use RIPA buffer as it denaturing and may be too harsh on your protein complexes. I would suggest using a non-denaturing lysis buffer. For example:
20 mM Tris HCl pH 8
137 mM NaCl
1% Nonidet P-40 (NP-40) (Triton X-100 can be substituted for NP-40)
2 mM EDTA
Store up to 6 months at 4°C. Immediately before use add protease inhibitors.
As for your question regarding centrifugation, as shown on https://www.abcam.com/index.html?pageconfig=resource&rid=11385#d, section 4.2 "Method B", step 5, we suggest centrifuging at 1,000-3,000 g for 2 minutes at 4°C.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
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Answered on Nov 09 2012