I am using Bacillus subtilis as a source for cellulase production grown on 0.1% CMC (Carboxy-Methyl-Cellulose) media. And now I want to detect the presence of cellulase using 0.1% congo red stain in a zymograph.
Hello I have been following this protocol for Endoglucanase screening and it does work for me all the time . give a try. and if your particularly looking for endo or glucosidases u can also try using Methyl umbelliferyl conjugated cellibiose which can be comparably easy to detect in SDS -PAGE by just U.V exposure.
Heres the protocol for congo red zymogram
Prepare 12% SDS-PAGE containing 0.5% CMC (low viscosity, Sigma: C5678). Keep it at 4ᵒC in running buffer for at least 2 h.
Ø Mix protein with 2X SDS Gel loading buffer (Without reducing agent like DTT or β-mercaptoethanol and do not denature by heat), keep at 4ᵒC for at least 1 h.
Ø Run the gel at 4ᵒC (in cold room). After electrophoresis separate gel from the cassette and incubate in isopropanol (20% in Phosphate buffer saline, PBS) for 2 min.
Ø Wash the gel 6 times with PBS (10 min each time).
Ø Incubate gel at 37ᵒC in PBS overnight.
Ø Stain with 0.1% congo red dye at RT for 30-60 min.
Ø De-stain with 1 M NaCl at RT for 15-30 min.
Ø Discard de-staining solution, add water or glacial acetic acid (100 µl in 50 ml water) and document it.
If you are doing it for endoglucanse then the protocol using 0.5% CMC will be better. this can be done by two methosds:
1. Incorporating CMC into the separating gel and after native PAGE incubate the gel at 50 Degree celsius
or
2. Do the native page and then overlay the gel with agar or agarose containing CMC and then incubate.
for visualization of halo zone you can either incorporate congo red into the gel/agar/agarose or you can food with congo red and NaCl for destaining.
you may also follow my article and modify it for your Native PAGE:
https://www.researchgate.net/publication/265847016_Simultaneous_Isolation_and_Screening_of_Cellulolytic_Bacteria_Selection_of_Efficient_Medium
Article Simultaneous Isolation and Screening of Cellulolytic Bacteri...
During the preparation of SDS PAGE only incorporate 0.2% Sodium salt of CMC'ase in the resolving gel and run the PAGE. Boil the samples and load on the wells. After running renature your enzyme by immersing the gel in Sodium succinate buffer (pH-5.8) containing 1mM DTT . The renaturation time may vary from 3 hours to 6 hours and then after washing gel it water incubate your gel in appropriate buffer for cellulase to act on CMC'ase at appropriate temp. Replace it with 0.1% Congo Red for atleast 10 min and then wash with 1M NaCl (3-5 min), Against the red background you will see white bands. to make these bands more visible you can rewash with water and add 5% acetic acid to get a bluish purple background with bands in white color.
We have been repeatedly getting very good bands with this procedure with Paenibacillus.
Good luck for your research.
u have to follow 3 mportant thngs while doing zymography,first maintain cool condition,second avoid heating....fixing of gel....all the best
Hello I have been following this protocol for Endoglucanase screening and it does work for me all the time . give a try. and if your particularly looking for endo or glucosidases u can also try using Methyl umbelliferyl conjugated cellibiose which can be comparably easy to detect in SDS -PAGE by just U.V exposure.
Heres the protocol for congo red zymogram
Prepare 12% SDS-PAGE containing 0.5% CMC (low viscosity, Sigma: C5678). Keep it at 4ᵒC in running buffer for at least 2 h.
Ø Mix protein with 2X SDS Gel loading buffer (Without reducing agent like DTT or β-mercaptoethanol and do not denature by heat), keep at 4ᵒC for at least 1 h.
Ø Run the gel at 4ᵒC (in cold room). After electrophoresis separate gel from the cassette and incubate in isopropanol (20% in Phosphate buffer saline, PBS) for 2 min.
Ø Wash the gel 6 times with PBS (10 min each time).
Ø Incubate gel at 37ᵒC in PBS overnight.
Ø Stain with 0.1% congo red dye at RT for 30-60 min.
Ø De-stain with 1 M NaCl at RT for 15-30 min.
Ø Discard de-staining solution, add water or glacial acetic acid (100 µl in 50 ml water) and document it.
We are using samples dissolved in sample buffer w/o DTT and not boiled. The samples are separated in 12 % SDS- PAGE. After electrophoresis, gel is incubated 5 min. in citrate buffer pH 5.0 (3 times) and finally incubated in a portion of MUC (previously dissolved in DMF) in the same buffer. After 15-30 min incubation, gel is UV exposed.
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