western blot using culture media - (Mar/09/2010 )

hi,

i am currently trying to do western blots with culture media for proteins secreted into it. We use really small gels with a 3-4ul well capacity so i have been lyophilising the media samples then resuspending in 1.5ul and adding 1.5ul loading buffer. I have not seen any bands yet though and i know from mass spec that the protein i am probing for is there.

80ul of media takes ~2hrs to lyophilise and the vacufuge temp options are room temp, 30, 45, and 60 degrees. I use room temp, but after 2hrs the samples are hot to the touch. Is there a different method to lyophilise the protein? I don't know if the protein concentrating methods will reduce the volume enough to allow me to load the whole sample into 1 well.

Thanks

rachel

Do you have an antibody that will detect the native epitope? Maybe you can IP your protein of interest out of the medium.

i will check the antibody and ask my supervisor if IP would be an option for us. Thanks

lab rat on Mar 9 2010, 09:46 PM said:

The antibody has been tested for IP, but my supervisor has said no to IP as it will be too difficult. Will try freeze drying rather than vacufuging and see how it goes.

Thanks for the help

rachelg on Mar 9 2010, 08:55 PM said:

i will check the antibody and ask my supervisor if IP would be an option for us. Thanks

lab rat on Mar 9 2010, 09:46 PM said:

Do you have an antibody that will detect the native epitope? Maybe you can IP your protein of interest out of the medium.

you can use spin ultrafiltration (eg microcon) to concentrate your samples. lyophilization and vacufuging will also concentrate medium components that may interfere with electrophoresis.