3 replies to this topic

[saad]

Hi All
I have some enquiries:
- I use In vitro translation to express the flag-prey and bait proteins "labeled with 35S-met" to test interactions.
then I expose on phosphor screen and scan using Typhoon machind, but the problem is our machine down for long time.
Could you help me find alternative methods to detect interactions where i deal with mammalian proteins.
I need sensitive methods .
Thanx

[mastermi]

There are several other methods for the detection of protein-protein interactions like

- Far Western (blot protein A on membrane, use tagged protein B as first antibody, use tag-antibody as second antibody (eg HIS))
- Co-Purification (bind tagged protein A on a column, put protein B on the column, wash and elute and see wether protein B is washed out or elutes together with protein B )
- Native PAGE (incubate protein A with protein B and both proteins alone, put the samples on a Native Gel, see wether complexes are formed between A and B )

Edited by mastermi, 07 June 2009 - 01:07 PM.

[saad]

Thanks a lot for your help
I mean also alternative methods to detect radioactive proteins.

[mdfenko]

you can use traditional autoradiography, that is, expose it to film. if you don't saturate the film then you can quantify the spots by scanning and integrating (you can use imagej to do this).