hello. I am a graduate student making recombinant proteins at university. Once the recombinant protein expression was successful.
Additionally, I plan to proceed with affinity chromatography and refolding using Ni-NTA.
So, I am making a Lysis buffer recipe, and the composition is as follows.
lysis Buffer Ingredients 20 mM Tris-HCL 0.5M NaCl 5 mM Imidazole 6M Guanidine hydrochloride 1 mM 2-mercaptoethanol NaCl (up to pH 8.0)
In addition, I would like to add the following reagents.
0.1% NP-40 (cell membrane degradation) 2mg Pepstatin (Protease inhibitor) 10mM Leupeptin (Protease inhibitor)
Will there be any problems with function and action even if I add additional reagents?
Dear Min Cheol Kim I would recommend adding a Halt Protease Inhibitor Cocktail to RIPA Lysis and Extraction Buffer before use.
Hi,
Adding those things should not be a problem, but they may not be necessary. Have you tried the lysis buffer without the reagents that you mentioned? Simpler is usually better and the more things you add to your buffer, the greater the chance of an unfavorable interaction.
If you include 6 M guanidine-HCl in the lysis buffer, the protein will be denatured. The only reason to do this would be if your protein is being expressed as inclusion bodies and you need to dissolve them and get the protein into solution. After purification, you would have to refold the protein, which is difficult. If the protein is being expressed in a soluble form, do not include denaturants such as guanidine.
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