bacteria inhibiting Staphyllococcus aureus - what to do more (Sep/04/2010 )

helloooo
I have a gram negative bacteria inhibiting S.aureus
I am precipitating its protein by ammonium sulphate. then I plan to run SDS.
What more can be done regarding this??
please suggest

You want to identify the gram negative bacterium or?

Hellooo
Not identificatioon of gram negative bacteria but about its product that is inhibiting S.aureus

Hola, I think that you want isolate the active product. Think,that it couldn´t be a protein, so first i would assay the growth extracellular medium in the case that your product was secreted. Antibiotics of protein structure like bacteriocins are little peptides so after sonication or any type of cell disruption I would made at least two step ammonium sulphate pptation, first untill 60% were big proteins will precipitate and afterwards to 100%, but I would assay the supernatant of 100% because the activity remains soluble. But probably my first choice were a ion exchanger column ( or making it in batch ) were according to its charge the compound will bound or not and could be eluted separatelly of other proteins. Good luck and have success.

hello protolder
i checked my supernantant with ammonium sulphate. 40, 60 and 80 percent. but did not get any precipitate at 40 and 60. at 80 there were precipitates but are very difficut to centrifuge. so i m confused hot to get them extracted.
rest when dissolved ppt in buffer they gave activity against S.aureus
problem for now is to extract all ppt then will go for SDS
rest u suggest..
thanks

Hola Arvind, I have understood that you have all your activity in the cold 80% pptate and you want analize by PAGE. I don´t know the apparatus that you have, but you need to separate well the precipitate so you have to increase rpm and time and do it at 4ºC. Afterwards, disolve it in buffer and dialyze to eliminate the excess of am. sulphate or you will have distorsinated the PAGE. Other irreversible method only for analysis would be precipitate the medium with 10% TCA ,suspend in water, add loading sol. (it turns yellow ) and 2-5 ul of 1M tris base untill it turns blue. Thinking on it, you could try precipitate the activity at acidic pH about 5, 4,5 and resuspend the pellet in buffer, checking that you have get near neutral pH. But you need a centrifuge able to get 15000-20000 g. I don´t Know the volumes that we are talking but you can use a table minifugue and collecting the pellets of some tubes. Good luck

Hellooo
thanks for suggestions. I have plans to dialize it. I have run simple native gel but never SDS so will seek help again.
I will do it after few days as I have to attend a workshop next week. so will come to topic after few days.
thanks...