Can someone give me a detailed protocol of sample preparation from cell line for zymography study. Addition of protease inhibitors, speed vac to concentrate the samples, bradford for estimation etc. in detail.
zymography protocol of millipore -search in google ........... that will give u good result
Zymography protocol:
Methods in molecular biology (Clifton, N.J.) 01/2012; 878:121-35
Dear Saba,
You can certainly follow the protocols shared by other contributors. I believe all are more or less similar and you will have a basic idea to begin with for zymography using cultured cells. I worked alot with MMPs and TIMPs for more than a decade and you can consult some relevant papers on that area through my RG page. These papers have been shared.
The basic points to be remembered are
1. First determine the localization of the enzyme (whether secretory or cellular). That will determine whether you need to work with the cell lysate or the culture media. Most often MMPs are secretory and concentrating the culture media my several folds (>20 times) will give a better digestion area in the zymography. I use either Millipore spin columns (10 kDa or 30 kDa) for such concentration purpose.
2. Serum free cell culture media or the cell lysate are the best choice. To starve the cells in serum free condition people use Insulin Transferrin Selenite (ITS) to keep the cells healthy for longer duration.
3. Never use any protease inhibitors for MMPs and EDTA or EGTA that can potentially chelate bivalent metal ions and calcium that are required for enzyme activity.
4. Depending upon the nature of the enzyme proper protein substrate (Gelatin, casein etc.) should be chosen in proper percentage.
5. Never heat, boil and reduce the samples with beta ME/DTT and ensure the gel run to be done in cold room (2-4 deg C) or the gel apparatus covered with ice to control the temperature.
6. After the gel run is over adequate precautions need to be taken for complete SDS removal with repetitive washing with 2.5% Triton-X100.
7. Careful preparation of calcium assay buffer (CAB) needs to be done as that will be used as incubation buffer for enzymatic digestion of the substrate protein in the gel over time. pH optima and duration of incubation needs to be determined carefully to optimize the enzymatic activity. Usually the temperature is kept constant at 37 deg C for zymogram assay.
8. Proper staining with Coomassie blue (2.5%) needs to be performed with constant monitoring. If there is an over stain controlled destaining with Acetic acid: Ethanol: Water (1:4:5) needs to be performed to get a better contrast for the digested bands for documenting the data.
Taking care for these small important points will certainly help you mastering over this technique.
Good luck!!
you just wrote everything I was looking for...thank you very much for such an elaborate reply...:)
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