Protein complex

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Harshal
Harshal's picture
Protein complex

Hello,

Here's my problem. I ran human diabetic kidney extracts on a 7.5% native gel to test whether my protein is complexed with other protein or not. the result showed that my protein was forming complex with collagen type I and fibronectin. I showed the result to my PI and he questioned that how do i know if the complex ( my protein- collagen tpye I and fibronectin) was formed in vivo and not after making kidney extracts. So can anyone tell me is there a way to test whether the complex is formed in solution whlile making extracts or its formed in vivo which is what i am expecting...plzzzzz help!!!

g a
g a's picture
Hi hardball

Hi hardball

You can use FRET determine that but the experiment then shall be performed on the cells rather than extracts.

eiorns
eiorns's picture
You can also image the

You can also image the complexes in tissue sections using confocal microscopy to determine if they are interacting in vivo. You can outsource your staining and imaging to a core facility at a top US university for free at http://thebenchhq.com. It's likely to be more cost effective than optimizing your experiment yourself.

Sami Tuomivaara
Sami Tuomivaara's picture
Harshal,

Harshal,

For FRET, you need fluorescent protein conjugates. Another possibility is to use two-hybrid assay. Both of these approaches are quite work-intense and require you to do molecular cloning.

Maybe a bit less effort-intense way is to do protein cross-linking experiment.

In any case, as Argerine pointed out, for in vivo work you need to have suitable cell culture line to do these experiments, kidney extract can't be used.

Cheers,