11 replies to this topic

[AbhayGene]

Hi all..
If I want to see the expression profile of a protein from log phase till stationery phase (at different time points) using western blot, should I dilute the samples so that all of them have same OD? I wonder if I directly do western blotting at different time points then increase in intensity of the band is due to increase in cell number. My objective is to see if the protein of my interest is getting expressed 'more' during stationary phase. I have been getting good suggestions from you guys. Thank you loads


Abhay

[gfischer]

AbhayGene, on Mar 24 2010, 04:42 AM, said:

Hi all..
If I want to see the expression profile of a protein from log phase till stationery phase (at different time points) using western blot, should I dilute the samples so that all of them have same OD? I wonder if I directly do western blotting at different time points then increase in intensity of the band is due to increase in cell number. My objective is to see if the protein of my interest is getting expressed 'more' during stationary phase. I have been getting good suggestions from you guys. Thank you loads


Abhay

I'd say either do protein isolation from a defined number of cells or use an identical amount of protein from each isolation by bradford.

[fishdoc]

AbhayGene, on Mar 24 2010, 04:42 AM, said:

Hi all..
If I want to see the expression profile of a protein from log phase till stationery phase (at different time points) using western blot, should I dilute the samples so that all of them have same OD? I wonder if I directly do western blotting at different time points then increase in intensity of the band is due to increase in cell number. My objective is to see if the protein of my interest is getting expressed 'more' during stationary phase. I have been getting good suggestions from you guys. Thank you loads


Abhay

Use a reference protein (DnaK or something) to standardize it for cell number. As the cells increase, so should the intensity of your reference band. Normalizing by CFU numbers and/or protein load is also useful. If you're planning on doing anything for publication, my guess is that any reviewer will want to see the internal reference done to verify the intensity of your target protein is greater or less than that of your reference as time passes.

I'm doing something similar now. I'm culturing bacteria in different conditions, so there is differential growth. I'm trying to pellet about 10^8 bacteria, then resuspend and lyse in a known volume of SDS-PAGE buffer. The CFU equivalents per ul of sample are somewhat normalized that way, and then I probe all of my westerns with anti-DnaK to ensure similar protein loads are in each lane. If the reference band is similar in all conditions, but the target band increases or decreases in intensity, I know expression is changing.

[pDNA]

the best way would be determine the cell dry mass, since OD-readings are heavily influenced by e.g. inclusion bodies formation ...after that use the same amounts of biomass for the SDS-PAGE (we use 1  mg i think) ...a reference protein is a great thing as well!

Regards,
p

[Prep!]

wesern will still be approximate only... and as western saturates at low amounts of protein u might not be able to get a proper dose response... why dont u try an ELISA? it can give u quantitative results....
if WB is the only option u have.. loading the sample from same number of cells will give u good approximation as others have stated too!!!

Best luck!!!

[AbhayGene]

Thank you guys...good piece of info. I have decided to go for Bradford assay as I dont have Dnak antibody if any further suggestions, welcome.


Thanks again.

[AbhayGene]

I am pinging again I dont know in which buffer should I lyse my cells pelleted. I am going to use Bio rad reagent for Bradford assat. But I dont know which is the ideal sample buffer for bacterial culture. kindly HELP!  

Thanks guys.

[fishdoc]

AbhayGene, on Mar 25 2010, 04:13 AM, said:

I am pinging again I dont know in which buffer should I lyse my cells pelleted. I am going to use Bio rad reagent for Bradford assat. But I dont know which is the ideal sample buffer for bacterial culture. kindly HELP!  

Thanks guys.

The Bradford Assay (and the Bio-Rad protein assay) can be problematic with lysis buffers as it is not compatible with a lot of detergents, which are used for lysis. You either need a different type of assay (BCA, etc) or use sonication instead of chemical lysis for whole cell lysate preparation. The sonication buffer can be formulated to contain low or no detergent (we've frequently simply used water for sonication before). After sonication is complete, the lysate is usually compatible with the Bradford Assay (or any other protein concentration assay available).

With that said, I will strongly recommend that if you plan on publishing any of this information, you evaluate potential loading controls for western (DnaK or others). I can't think of any western data I've seen in a paper that didn't have corresponding information for a reference protein, particularly if you're trying to show up or down regulation of something.

[AbhayGene]

OK Fishdoc...Thank you so much!
Now, my question is how long the sds-page buffer lysed sample is stable in -20?
As I was away for some reasons, I lysed the samples in sds-page buffer,cooled and placed them in -20 for more than 10 days. Can I use those samples for western blot now? are they stable enough?

fishdoc, on Mar 25 2010, 07:12 PM, said:

AbhayGene, on Mar 25 2010, 04:13 AM, said:

I am pinging again I dont know in which buffer should I lyse my cells pelleted. I am going to use Bio rad reagent for Bradford assat. But I dont know which is the ideal sample buffer for bacterial culture. kindly HELP!  

Thanks guys.

The Bradford Assay (and the Bio-Rad protein assay) can be problematic with lysis buffers as it is not compatible with a lot of detergents, which are used for lysis. You either need a different type of assay (BCA, etc) or use sonication instead of chemical lysis for whole cell lysate preparation. The sonication buffer can be formulated to contain low or no detergent (we've frequently simply used water for sonication before). After sonication is complete, the lysate is usually compatible with the Bradford Assay (or any other protein concentration assay available).

With that said, I will strongly recommend that if you plan on publishing any of this information, you evaluate potential loading controls for western (DnaK or others). I can't think of any western data I've seen in a paper that didn't have corresponding information for a reference protein, particularly if you're trying to show up or down regulation of something.

[AbhayGene]

OK Fishdoc...Thank you so much!
Now, my question is how long the sds-page buffer lysed sample is stable in -20?
As I was away for some reasons, I lysed the samples in sds-page buffer,cooled and placed them in -20 for more than 10 days. Can I use those samples for western blot now? are they stable enough?  

[mdfenko]

AbhayGene, on Apr 8 2010, 12:15 AM, said:

OK Fishdoc...Thank you so much!
Now, my question is how long the sds-page buffer lysed sample is stable in -20?
As I was away for some reasons, I lysed the samples in sds-page buffer,cooled and placed them in -20 for more than 10 days. Can I use those samples for western blot now? are they stable enough?  

they should be fine. you may have to reboil. just ensure that there is no sds out of solution.

[AbhayGene]

mdfenko, on Apr 16 2010, 11:54 PM, said:

AbhayGene, on Apr 8 2010, 12:15 AM, said:

OK Fishdoc...Thank you so much!
Now, my question is how long the sds-page buffer lysed sample is stable in -20?
As I was away for some reasons, I lysed the samples in sds-page buffer,cooled and placed them in -20 for more than 10 days. Can I use those samples for western blot now? are they stable enough?  

they should be fine. you may have to reboil. just ensure that there is no sds out of solution.

Alright. Thanks