Trying to isolate a recombinant protein in bulk amount so it would be best if a large volume of lysis buffer can be made in lab.
There are a lot of things to consider for a lysis buffer. The type of cells you are trying to lyse as well as the identity of the protein. Is this done in E. coli, yeast, Xenopus, or insects? Also, what are the characteristics of the protein; is it phosphorylated, hydrophobic, aggregate, lipidated, glycosylated? These characteristics will determine the type of detergent you should use (ionic, non-ionic) and the concentration.
Most lysis buffers use hypertonic or hypotonic solutions to drive the water out of or into the cells. Detergents then break up the cells, and then proteases and phosphatases need to be inhibited. Free cations are chelated to deactivate some enzymatic processes.
The simplest buffer I know is NET lysis, which is NaCl (150 mM), EDTA (50 mM) and Triton-X100 (100 mM) supplemented with a protease cocktail inhibitor, but with out knowing your system or protein, there is no guarantee it will work.
This is a tough question without further information, so I assume you are just starting out. I found it helpful to start with the lysis buffers recommended in the QIAexpressionist (50mM phosphate buffer pH8, 300mM NaCl, 5-10mM imidazole in case you are using a 6xHis-Tag). Of course, it is helpful to adapt the buffer to your specific needs for which further information is definitely needed. Higher salt concentration (e.g. 500mM) or adding detergents might help you with precipitation (OG, Tritonx-100, NP-40). I've also added DTT to lysis buffers before hoping to stabilize my proteins. Anyway, all these buffers and suggestions can be made in a lab in bulk.
Link to the QIAexpressionist:
http://www.qiagen.com/resources/Download.aspx?id=79ca2f7d-42fe-4d62-8676-4cfa948c9435&lang=en&ver=1
There are a lot of things to consider for a lysis buffer. The type of cells you are trying to lyse as well as the identity of the protein. Is this done in E. coli, yeast, Xenopus, or insects? Also, what are the characteristics of the protein; is it phosphorylated, hydrophobic, aggregate, lipidated, glycosylated? These characteristics will determine the type of detergent you should use (ionic, non-ionic) and the concentration.
Most lysis buffers use hypertonic or hypotonic solutions to drive the water out of or into the cells. Detergents then break up the cells, and then proteases and phosphatases need to be inhibited. Free cations are chelated to deactivate some enzymatic processes.
The simplest buffer I know is NET lysis, which is NaCl (150 mM), EDTA (50 mM) and Triton-X100 (100 mM) supplemented with a protease cocktail inhibitor, but with out knowing your system or protein, there is no guarantee it will work.
Hi,
I would prefer you to add enzymes(DNase1 and Lysozyme100µg/ml each) and Protease inhibitor cocktail - EDTA-free, where the lysis will be effective to extract the protein and Phosphate buffer should Di basic.
Enzymes are more sensitive, so it is prefer to prepare the buffer prior to the lysis, as well the stability of the enzyme in buffer at 4°C is around approx 2weeks.
Hope it works to isolate the protein
Goodluck:)
i want to ask for lysis buffer for tissue.THIS before DNA EXTRACTION or after
I am trying to isolate a protein having hydrophobic moieties from E coli cells. The protein will be expressed after giving overnight inoculation in a flask and isolated from the E coli culture. Thank you all for your excellent suggestions!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques