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

 

[ravenprp]

Hi Nautica,

Thank you for sharing your findings on the sequence in fibronectin that attaches to the fibronectin receptor. The RGD sequence is indeed a well-known binding motif that is found in many extracellular matrix proteins, including fibronectin. This sequence has been shown to bind to a specific integrin receptor on the cell surface, which is responsible for mediating cell adhesion and signaling.

To use this information to isolate the fibronectin receptor, you can design an experiment where you use a synthetic peptide with the RGD sequence to compete for binding with the fibronectin receptor. By adding the synthetic peptide to a cell culture, you can block the binding of fibronectin to the receptor and therefore isolate the receptor. This approach is commonly used in studies to identify and isolate specific receptors.

Additionally, you can also use antibodies that specifically target the integrin receptor to isolate it from a cell lysate or tissue sample. These antibodies can be immobilized on a solid support, such as a column or plate, and used for affinity purification of the receptor.

I hope this helps and provides some ideas for your further experiments. Best of luck with your research!