Please tell me can i confirm that my protein being expressed by simple checking OD@ 600 of induced and uninduced culture?
If yes what will be the difference in OD between induced (with IPTG) and uninduced culture before and after induction.
OD600 follows the increase in cell density of your bacterial cell culture, and therefore induced and uninduced cultures should not have any significant difference that could be an indicator of successful protein over-expression. However, in some cases, the protein being over-expressed can alter cell growth, e.g. in cases where there is strong protein aggregation or the target protein being known as toxic for bacterial cells. In these cases, a lower absorbance at OD600 compared to an uninduced cell culture can be expected.
The best way to check protein over-expression, in my humble opinion, remains to be SDS-PAGE. You might want to try a small-scale over-expression protocol, using a small cell culture and taking aliquots of ~10 mL every X amount of hours after IPTG induction (e.g. taking cell culture samples every 2 hours after induction) and then disrupt the bacterial cells in these aliquots and perform an SDS-PAGE with the cell extract. By doing these, you might be able to identify whether your protein is being over-expressed or not.
Good luck!
OD600 follows the increase in cell density of your bacterial cell culture, and therefore induced and uninduced cultures should not have any significant difference that could be an indicator of successful protein over-expression. However, in some cases, the protein being over-expressed can alter cell growth, e.g. in cases where there is strong protein aggregation or the target protein being known as toxic for bacterial cells. In these cases, a lower absorbance at OD600 compared to an uninduced cell culture can be expected.
The best way to check protein over-expression, in my humble opinion, remains to be SDS-PAGE. You might want to try a small-scale over-expression protocol, using a small cell culture and taking aliquots of ~10 mL every X amount of hours after IPTG induction (e.g. taking cell culture samples every 2 hours after induction) and then disrupt the bacterial cells in these aliquots and perform an SDS-PAGE with the cell extract. By doing these, you might be able to identify whether your protein is being over-expressed or not.
Good luck!
OD of culture is the measure of cells. You can not use that as a measure of protein present in the cells. To see if the induction worked, make cell lysate of both uninduced and induced cultures, equalize the protein concentration and run SDS page. You can also do western (if the antibody is available) to make sure that induced protein indeed is your protein of interest. However you might need to standardize the conditions for induction.
If your protein forms inclusion bodies in the cells, this can affect how much light gets blocked by each cell. So I vote yes :) And the amount of difference, it depends on the nature of the inclusion bodies...
Theoretically NO, unless you are expressing a protein that is (1) toxic to bacteria it self, and thus killing cells (not allowing further growth) upon stronger induction or (2) or you are expressing a protein that has a chromophore such as heme binding (red colored cells!) thus creating a OD 600 (visible range) interference! best wishes.
there is no detectable difference. maybe uninduced bacteria grew a little more from induced bacteria, because induced bacteria use most of their energy for expressing.
so It's not a good mean (od=600) for studying expression
Adding to Fereshteh's and previous people's nice answers, you have to keep in mind that it is a certain stress for bacteria to express something that they do not have endogenously. But it depends on the size and complexity of the protein that you want them to express. As a rule of thumb, the bigger it is, the more stress it may be for the poor guys (but it's also about toxicity as was mentioned); so you may have to find out the optimal time (as suggested by Cesar), the optimal IPTG concentration and optimal temperature of induction.
Theoretically no,
but if you start the expression during the log phase (sufficiently, before the stationary phase), the rate of increase in OD will decrease as compared to a non-induced one; this can be an operational (experimental) sign.
Hi there,
The possible effect of protein expression on cell growth is dependent on the nature of the protein expressed then you can't make a general statement. To answer the question : it's yes if expression affects growth and no if expression doesn't affect growth...
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