Abstract
This protocol is used to purify His-tag ubiquitin conjugated protein. In this particular case, cells were transfected with His-tag ubiquitin and p53 which allowed us to purify using His-tag and reveal the WB using antibodies against p53 to see just the p53-ubiquitinate. The present protocol can be used in general for His-tag proteins expressed in mammalian cells.
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was adapted from Gajjar et al. (2012). This work was supported by INSERM, La Ligue Contre le Cancer, and RECAMO CZ.1.05/2.1.00/03.0101. M.M.C. was supported by JSPS, AXA Research Fund, and Fundacao para a Ciencia e Tecnologia of Portugal. M.G. was supported by a research fellowship from Indo-French Centre for the Promotion of Advanced Research (IFCPAR) and from the ARC.
References
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Hello!I think its depend on the endogenous levels of your protein. Using MG132 you will have an accumulation of your ubiquitinated protein, then it is possible, but you have to try! good luck!
HelloI tried a couple of times, and I could see the overexpression but when the proteins are small, but if your protein is big, it will be more difficult. I hope this could help you.Vanesa
Thank you, I will try both approaches :)
Hello XiaoIndeed we used just one 76 aa coding gen, and the cell add an unknown number of ubiquitin His tagget to the protein. I hope this information helps to answer your question.Vanesa
Thank you, Vansesa
Hello, the pH should be 8
Thanks Vanesa,Let me make sure for the Lysis buffer: the pH should be the same as Tris-Hcl? so the 0.1M Na2HPO4/NaH2PO4 pH should be 8 in Lysis buffer and 6.8 in Wash buffer? or both are the same pH:7.0? Please confirm.Thank you again.
yes, indeed for lysis buffer pH should be 8 and for wash buffer should be 6.8. Hope you have a nice experiment!!
yes! indeed is 75ul
Thanks for your reply
yes you are completely right! the good concentration is 10mM, im sorry for this mistake.On the other hand 5ml is the volume that for my proteins gave the best yield
Per author's request, the concentration of Tris-HCl has been corrected to 10mM in the protocol.Sincerely,Bio-protocol editing team
Hello and sorry for the delay in the answer!!. Yes you can keep 15-30ul of the lysate.cheersVanesa
When pH > pI, a protein has a net negative charge. When pH < pI, a protein has a net positive charge. In the higher pH of lysis buffer(8.0), your His-tagged protein binds to the Nickle beads strongly, but at the same time some nonspecific proteins are negatively charged and bind to the Nickle beads. In the lower pH of washing buffer (6.8), your His-tagged protein still remain on the beads with strong affinity due to its His tag. The nonspecific proteins bound to the beads have less (if not zero) negative charges and dissociate from Nickle beads. So after washing step, the nickle beads should only bind to the His-tagged protein.
I am not the author, but let me try to answer your question: I would think it is commonly accepted that the pH is the Tris pH. As a result, the buffer ends up in the same (or similar) pH as Tris. But it is a good lab practice to always check pH of the buffer especially when it is your first time to make such buffer. Another caution is that samples you will add in the buffer does not maximize its buffer capacity, which is also done through sensibly checking pH.
Hello and sorry for the delay in the answer.Im agree with Lin, is commonly accepted that the pH is the Tris pH. Just one point, in the case of wash buffer you have also the component of phosphate buffer around 8, but you slowly will go down the pH (because of the explanation of Lin) of each buffer to improve the purification.