How Can I Use the RGD Sequence to Isolate the Fibronectin Receptor?

[nautica]

I have found the a.a. sequence in fibronectin that attaches to the fibronectin receptor.

I have concluded RGD. How can I use this to isolate the fibronectin receptor?

Thanks
Nautica

 

[iansmith]

You could use affnity Chromatography isolation.
http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/WoodW/affchrom1.html

Or a process derive from affinity Chromatography which can be done in a tube.
1- You attach the ligand to either to biotyne or agarose beads.
2- You mix the potential receptor with the attach ligand and incubate in a solution and at a temperature that is good for binding.
2b- solubilize the protein if it is a membrane protein. This could also occur before step 2.
3- You then treat with low salt solution to remove unspecific binding, you centrifuge and remove the supernatant and keep the pellet, and treat and centrifuge again until you think you have mostly only the desired receptor.
4- Then you treat with high salt solution and other steps to remove the binding and centrifuge but in this case you keep the supernatant.

This is the steps we use to isolate Hb-binding receptors
http://www.geocities.com/nivenlab/Hb_AI.pdf