Hello,
When I grow two newly isolated bacterial strain which are silver resistant on a LB agar plate, I am noticing a zone of clearing around the colonies. These zones of clearing are Ag free.
According to previous research, zones of clearing which push away a substrate are usually caused by some exoenzyme, or in some cases, a chemical.
I want to see if I can isolate this exoenzyme from the agar plate. I know that I will have to use some form of PAGE electrophoresis. However I am not sure exactly how to extract and purify the protein from the LB agar.
Does anyone know of any method for doing this?
Thanks.
Why would you not try to purify from a liquid culture. The amount of protein/chemical secreted around a single colony is likely to be very very small, this would be the equivalent of trying to purify a protein from 100ul of culture.
Why not grow a 1liter or so liquid culture? If you think the factor requires Ag to have expression induced, then add low levels of Ag to the liquid culture.
Do you have a good assay or means of detecting the activity of what you are looking for?
A few possibilities:
1. Rather than using normal agar, redo your experiment using agarose.
2. Instead of using LB, try defined (for example for E. coli use supplemented M9).
Both of the above two steps will help you with starting material that may be considerably cleaner and also making it easier to isolate protein(s) of your interest.
Depending on the diameter of the circle, you may be able to excise the clear agarose as a plug, wash all the bacteria away, accumulate sufficient plugs to pool the material.
Pour a new horizontal gel with agarose, create a barrier with a dialysis membrane and run all the proteins (at least anionic ones) against the dialysis membrane. The proteins will be dilute at this stage but you can concentrate them by any number of methods routinely used for protein purification.
If you have some knowledge about the heat stability of your protein(s) interest and if they stable at 40-45C, use sift agarose, which can be melted at low temperature - add an ion exchange material (such as DEAE Sepharose for anion exchanger) to the liquified material at low salt, dilute the suspension so it does not congeal at room temp, spin out the ion exchanger and it would have bound proteins,which can be eluted by raising salt concentration. Obviously, you will have to play around with conditions to optimize them.
What is the evidence for an actual secreted factor "pushing away" the silver? Isnt a more logical explanation the chelation (possibly with intracellular storage) analogous to siderophores? In other words, why are you expecting a protein to be present in the cleared zone?
@Weip Klaas Smits - This is also a possibility, and I am looking into it through electron microscopy. Its one of two things, some sort of accumulation as you described, or some sort of exoenzyme which is pushing away the silver. The zone of clearing is very large, which makes it less likely to be some sort of accumulation.
I have some pictures on what I am seeing.
I start from the last suggestion given from Tausif Alam:
"If you have some knowledge about the heat stability of your protein(s) interest and if they stable at 40-45C, use sift agarose, which can be melted at low temperature"
Once you have your excised circle from agarose and melted at 40°C, you may also try to load it directly into the well of a PAGE (with or without SDS, is up to you). I have not idea of how many different secreted protein there are or if you may have enough to be detected with silver staining on a PAGE, but it should be very fast to try and may be revealing...
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