I want to do zymography , for that i have seperated my cells and media,i've pelleted my cells and now i want to lyse it for zymography, can anyone please suggest me a good method for that so that i will get active proteins...
And also how do i precipitate active proteins from spent media? Help me please,,,,,,,,
Jinsha,
Generally you can do a simple lysis for zymography, but it does depend on your cell type and the enzymes you want to visualize and a bit on your gel system. You can lyse the cells by homogenization in a simple buffer, such as PBS or even distilled water, if you are not trying to maintain the lisate for a long time. Two volumes of buffer to one volume of cells is appropriate for many enzymes, but you need an appropriate homogenizer for the amount of cells you have.
A freeze-thaw system works for small samples. Use a hypotonic buffer, such as 10 mM Tris, pH 8.0, for example, and resupend the cell pellet well in a 1.5 ml microcentrifuge tue. Freeze it at -80. After it is solidly frozen, thaw the lysate, perhaps in a 37 C waterbath, so that it thaws completely and quickly. Vortex briefly, and return to -80. Do three freeze-thaw cycles, then centrifuge in a microcentrifuge to pellet the debris. Use the supernate to load your gel.
Some enzymes show better results by addition of a dtergent, such as 0.1 to 1% Triton X-100 to the lysis buffer. You want to make sure NOT to use a phosphate beffer if you will be using a Tris-glycine gel system, and the phosphate will interfere with stacking and cause smears. Lysates can be stored frozen for some time and retain activity of many enzymes, but that varies.
Precipitating active proteins form spent media can be tricky, as you may have many proteins there from serum and lysed cells. Standard ammonium sulfate fractionation can be useful, if you know the solubility of your enzymes. Many are known to be preciptated at a specific concentration range of ammonium sulfate, such as in a 35 to 50% range, meaning 35 to 50% of full saturation of ammonium sulfate in water. If you can find the reference range for your protein, you would mix the media with saturated ammonium sulfate solution to the lower final concentration, incubate on ice with gentle agitation, such as on a rocking platform, for 15 minutes, then centrifugefor 25 minutes at 15,000 x g to precitpitate. Take the supernate, add more ammonium sulfate solution or solid ammonium sulfate to reach the upper concentration, mix, and repeat the incubation on ice and centrifugation. The first step removes proteins that precipitate at lower concentrations and gives you a cleaner prep. You will still have a number of other proteins precipitating with yours, but it will be more concentrated when you resuspend the pellet in a small volume. Many enzymes are supplied commercially in ammonium sulfate slurries, so activity is usually not a problem.
I hope thisa helps and is not too late, since I have not checked this board in a while. Good luck!
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