To increase the sensitiveness in sensing of alkyl sulfatases in Native PAGE gels, we report here a simple staining method based on Coomassie brillent blue R 250. This method facilitates in sensing of swoon alkyl sulfatase sets which are otherwise hard to visualise.
Alkyl sulfatase, Colloidal coomassie, Polyacrylamide gel ( PAG )
Alkyl sulphate esters are known to possess first-class cleansing belongingss and are widely used as detergents. Sodium dodecyl sulphate is a member of the group and is one of the widely used detergents in families and Industry. Apart from its usage in family, SDS is besides used in many other applications ( Karsa, 1992 ) . It is good documented that SDS is toxic to animate beings and micro-organisms, particularly to fishes, which come in direct contact with this detergent as mainly it is disposed in immense sums in H2O organic structures. So, it is considered as a pollutant in H2O systems ( Rosety et. al. , 2001, Bantseev et. Al. 2003 ) . Due to its toxicity, it was realized that bioremediation of this detergent is of import. This initiated the showing and designation of bacteriums, which could degrade and metabolise SDS. Members of Pseudomonas were found capable of degrading this detergent and until now, most of the work on biodegradation of SDS is done on this bacteria. Pathway of biodegradation of SDS is initiated by cleavage of sulphate group from SDS, ensuing in the formation of 1- dodecanol ( C12 intoxicant ) . This measure is catalysed by the enzyme alkyl sulfatase. 1- dodecanol is so oxidized to 1- dodecanoic acid by intoxicant dehyrogenases. 1- dodecanoic acid is so metabolized via beta oxidization tract and used as a C beginning ( Thomas and White, 1989 ) . In Pseudomonas sp. , it has been reported that multiple alkyl sulfatases of different molecular weights and substrate specificities exist. Until now, merely two alkyl sulfatases have been to the full characterized. So, in work affecting biodegradation of SDS, it is indispensable to analyze the enzyme involved in biodegradation. Alkyl sulfatase can be easy detected by Native PAGE Zymography. Zymographic sensing of alkyl sulfatase involves incubation of PAG in a solution incorporating 20mM SDS in 0.1M Tris-Cl for 30 mins to 1-2 h. Alkyl sulfatase cleaves SDS and 1- dodecanol is formed, which forms a white precipitate in PAG. This method is simple but it is hard to visualise because many a times, these white sets are really weak and are unstable. They disappear rapidly in 2-3 H due to diffusion of 1- dodecanol in to the medium ( Ellis et. al. , 2001 ) . This limits in the word picture and designation of the enzyme. Since, for the designation of enzyme, involves alining the sets on the zymogram gel with matching protein sets on an tantamount gel without substrate. Sets are typically excised from the tantamount gel for protein designation by mass spectrometric analysis. Besides for molecular weight appraisal, the zymogram gel is aligned with an tantamount gel incorporating the molecular weight markers. Since, in the present instance, the alkyl sulfatase sets are really unstable and they disappear in 2-3 H due to diffusion of 1-dodecanol in the medium, so it is really hard to place the proteins with this method. So, we wanted to develop a method to forestall diffusion of 1- dodecanol into the medium. So that, the alkyl sulfatase sets originating from activity staining can be aligned with an equivalent gel which is stained with CBB staining. The corresponding set can be cut and analyzed by maldi tof analysis.
In the present survey, we report here a simple staining process for increasing the sensitiveness and thereby, sensing of the alkyl sulfatase sets by staining the sets with Coomassie brillent blue R 250. In this procedure, petroleum cell infusions of SDS adult cells belonging to P. aeruginosa strain SDS3 ( EF197939 ) ( Lane 1 ) , P. stutzeri strain K2 ( GQ328718 ) ( Lane 2 ) and P. alcaligenes strain JN2 ( GQ328720 ) ( Lane 3 ) were prepared harmonizing Ellis et. Al. ( 2001 ) and were run on native PAG incorporating 5 % Acrylamide in stacking and 6 % in deciding gel. The cataphoresis was conducted at 40C and 15 ma current, for minimising the heat inactivation of enzymes. After cataphoresis, the PAG gels were incubated in solution incorporating 20 mM SDS in 0.1M Tris-Cl at 370 C. Until, the sets were observed ( Figure 1A ) . These PAG were transferred to Colloidal coomassie staining solution for 4-5h. The fixing measure in 40 % ethyl alcohol and 10 % Acetic acid was omitted because it was thought that it would ease diffusion of 1- dodecanol in the medium. After incubation it was observed that, the white sets of 1- dodecanol were stained dark blue and the gel was light blue in colour ( Figure1 B and C ) . After staining the gel was taken out from the solution and washed 2 times with dual distilled H2O and exposure was taken. It was easy apparent that swoon sets matching to alkyl sulfatase which were swoon ( Lane 2 A ) and non clearly seeable were easy to place. The staining of sets by Coomassie brillent blue R 250 can be attributed to its high solubility in intoxicants instead than H2O. Since merchandise of SDS cleavage is 1-dodecanol ( C12 intoxicant ) . So, it is obvious that the dye has higher affinity towards alcohols as a consequence sets were stained blue. Besides, staining of sets with the dye blocks the diffusion of 1aa‚¬ ” dodecanol in the medium. Previously it was observed that when PAG were incubated in solution of SDS in Tris-Cl, the sets were seeable after 1 H of incubation and they disappeared after 2-3 H due to diffusion of 1-dodecanol into the medium but here after staining with the dye for 4-5h, the sets are still present. It can be concluded that staining of sets with Coomassie brillent blue R 250 increases the sensitiveness for the sensing of alkyl sulfatases in native PAG. This method is simple and efficient for sensing of Alkyl sulfatase by native PAGE Zymography.
Figure1.Zymographic sensing of alkyl sulfatases in native PAG. ( A ) Detection of alkyl sulfatases by incubation in 20mM SDS in 0.1M Tris-Cl, ( B ) and ( C ) Colloidal coomassie stained sets, ( B ) Image acquired by gel certification Unit, ( C ) Image acquired by Canon digital camera A530. Lane 1, 2 and 3 represent P. aeruginosa strain SDS3 ( EF197939 ) ( Lane 1 ) , P. stutzeri strain K2 ( GQ328718 ) ( Lane 2 ) and P. alcaligenes strain JN2 ( GQ328720 ) ( Lane 3 ) severally.