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I used to work with PAHs (phenanthrene, pyrene,...) and we disolved the compound in acetone. However if you added to a medium without a surfactant (I used Tween80), it appeared as cloudy due to thin precipitation but concentration was 100 uM in the media, 1000 times higher than yours. It also depends on your compound, medium,...
If you give details of compound, culture media,... would be easier to give a proper response
#140072 Drug recrystallize when i added to DMEM
Posted
on 27 August 2012 - 05:34 PM
#140081 Drug recrystallize when i added to DMEM
Posted
on 28 August 2012 - 12:54 AM
Guys, I merged your two questions because they are very similar (and came one after the other, please check the forum before posting).
Andreea
Andreea
#139331 Overexpressed protein shows a band higher than expected?!
Posted
on 12 August 2012 - 12:52 PM
It is very common for proteins not to run at the expected or predicted molecular mass. In denaturing systems, the overall charge of the protein still has some bearing on how it runs, despite the SDS and supposedly linear conformation.
It is also possible that the protein is post-translationally modified in some manner.
Antibody data sheets can also be a bit misleading - often they show bands that are against the antigen only, or against purified protein that was expressed in a non-native system (e.g. e. coli for a eukaryote protein).
It is also possible that the protein is post-translationally modified in some manner.
Antibody data sheets can also be a bit misleading - often they show bands that are against the antigen only, or against purified protein that was expressed in a non-native system (e.g. e. coli for a eukaryote protein).
#139765 Overexpressed protein shows a band higher than expected?!
Posted
on 22 August 2012 - 02:02 AM
I think the question is if with these antibodies you detect that the endogenous protein has a lower molecular weight band. If this is not the case and you never detect endogenous levels, it is not surprising to have differences between the stimate MW and the real one. To be sure, you can check the overexpression construct to make sure the sequence is correct.
If everything is OK and the endogenous protein does have a lower MW could be that, as bob1 suggested, overexpressed protein is having some posttranscriptional modification... however, I would try to do RNAi against your protein of interest, because maybe the antibodies are not sensitive and/or specific enough. I had a similar problem, but the difference between real and expected MW was too much to have doubts. The antibody only recognised overexpressed protein (single band) but when non-transfected cell extracts were used, the antibody recognised a non-specific band... Just to be sure I performed RNAi against that protein and confirmed the "endogenous" protein recognised by the antibody was not the good one...
If everything is OK and the endogenous protein does have a lower MW could be that, as bob1 suggested, overexpressed protein is having some posttranscriptional modification... however, I would try to do RNAi against your protein of interest, because maybe the antibodies are not sensitive and/or specific enough. I had a similar problem, but the difference between real and expected MW was too much to have doubts. The antibody only recognised overexpressed protein (single band) but when non-transfected cell extracts were used, the antibody recognised a non-specific band... Just to be sure I performed RNAi against that protein and confirmed the "endogenous" protein recognised by the antibody was not the good one...
