3 replies to this topic

[wingfly]

Hello everyone,
     I have some questions to ask you. I have run native-PAGE electrophoresis, and the sample is cell lysate. I got some bands in native-PAGE. Now,I want to identiy the components of the native bands. I know a method that need to transfer the native bands onto SDS-PAGE, after electrophoresis, using mass spectrometry to identify the bands/spots in SDS-PAGE. When I try this method, there are many trails following the bands in SDS-PAGE. Does anyone know how to eliminate or reduce trails? Or Does anyone  know other methods to analyze and identify the components in native bands?
      Any suggestions are to be grateful. Thank you very much.  

[mdfenko]

trails are caused by aggregates of the protein. you can reduce the trails by incubating with sample buffer for a longer period of time and/or elevated temperature. the problem with this is that you will lose more of your protein (it will diffuse out of the gel).

you should just ignore the trailing and cut out the spot at the end of the trail.

[wingfly]

mdfenko, on Oct 20 2009, 12:20 AM, said:

trails are caused by aggregates of the protein. you can reduce the trails by incubating with sample buffer for a longer period of time and/or elevated temperature. the problem with this is that you will lose more of your protein (it will diffuse out of the gel).

you should just ignore the trailing and cut out the spot at the end of the trail.

Many thanks for your suggestion. I will have a try. Do you konw other methods to identify the proteins  in one native band except transferring it onto SDS-PAGE? Thank you!

[mdfenko]

if you know what protein(s) you seek then you could immunoblot, in-gel enzyme assay, differential stain (for type of protein, such as calcium binding protein, glycoprotein, etc.) amongst other methods.