Hi
I am looking for a method/recipe for making a lysis buffer for extracting coloured proteins from E.coli K12. The coloured proteins are derived from GFP.
The antibiotic used is Chloramphenicol.
I am starting off learning the process with kits.
The lysis Accelerator is Hens Egg White Lysozyme and the Lysis buffer is Triton-x-100 mixed with some other things that the company cannot disclose to me.
Does anyone have any suggestions about making my own Lysis buffer from the triton-X-100?
For context I am a textiles design student working in a lab and my aim is to extract the coloured proteins/pigments and experiment with dyeing with them. I don't need the process to be perfect but I would like to efficiently extract the colour from the E.coli cells
many thanks
Ruth
Any lysis buffer is going to just break open the cells (hopefully) and release all of the proteins, not just the GFP-derived ones. After lysis, you will have to purify the proteins of interest. This will be expensive to do at a large enough scale to use the proteins as dyes. Moreover, the color is only retained if the protein is properly folded in its native conformation. Compared to organic dyes, proteins are usually rather delicate and unstable (damaged by heat, drying out, exposure to harsh chemicals or solvents or extremes of pH). Also, their molecular weights are much higher (~50-100-fold) than organic dyes, so the amount of protein needed to get as much color as you would get from organic dyes, in terms of milligrams, is much higher.
Hi,
thanks for the info :)
Yea I'm doing very small samples, I have some of the kits by Amino labs and they use the pigments to colour fabrics, I'm testing if I can cultivate slightly larger amounts and make printable pastes in very small quantities.
the lab I use has the kit to purify the proteins so only the colour remains which is great!
If anyone has a suggested recipe for the lysis buffer that would be greatly appreciated.
Amino se one with a triton-x-100 base by could not let me know the other ingredients as its a trade secret, they suggested I try another recipe.
I am expressing a protein that has 2 GFP derivatives at both N- and C-terminals. One of the fluorescent proteins has a chromophore that matures slowly, so we have to express the protein during 2 days with high shaking. Later we use a standard lysis buffer and we have lots of this protein. The lysis buffer contains 20 mM Tris-HCl, 300 mM NaCl, 20 mM Imidazole, 1 mM DTT.
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