2
$\begingroup$

Short summary

I'm having a glass surfaces and I want to adsorb proteins onto them. I have no problem, when I first adsorb fibronectin (human) onto some areas and afterwards any other protein. The second protein will only adsorb onto the remaining "free" areas.

But as soon as I apply fibronectin in the second place, it just adsorbs everywhere. And I want to avoid this.

More detailed Protocol

(direct microcontact printing):

  1. Incubate a pdms-stamp with Protein-Solution #1 for some time and concentration
  2. Dry pdms stamp and press onto coverslip for some time
  3. Remove stamp and put a drop of Protein-Solution #2 for some time and concentration onto the coverslip

I varried already concentrations and times without noticeable success. I also tried adding some BSA to Fibronectin - also without success. All solutions are within PBS.

Further steps I anticipate:

  1. Varying pH around the protein's isolectric point (Fibronectin: around 5.2)
  2. Adding some detergens to fn-solution (e.g. Tween)
  3. Variation of salt-content in fn-protein-buffer

My question

I'm wondering what the exact reason for the stickiness of Fibronectin is and which parameter is the best to vary? Did I miss something? Does Fibronectin stick to the other proteins or rather to "free areas" of the surface?

Please note: surface modification techniques (e.g. using epoxys) are no option.

I'm not sure, whether this topic perhaps suits the chemistry-community better. Feel free to move.

$\endgroup$
  • $\begingroup$ What exactly do you mean by "But as soon as I apply fibronectin in the second place, it just adsorbs everywhere."? Are you applying another protein (which one?) in spots, then flooding the coverslip with fibronectin? Fibronectin, like the related collagen proteins, is extremely "sticky" and will bind to many surfaces, as well as many different types of proteins. $\endgroup$ – MattDMo Sep 18 '15 at 20:33
  • $\begingroup$ Exactly, that's what I'm doing. I tried various other proteins (e.g. Laminins and Vitronectin). My idea is to modify the conditions when applying Fibronectin (e.g. pH), to make FN less sticky. $\endgroup$ – AnatraIlDuck Sep 21 '15 at 8:40
1
$\begingroup$

What is the exact reason for the stickiness of Fibronectin?

Fibronectin interacts with diverse substrates which reflects its non-specificiy and its interaction with hydrophobic substrates is better suggesting that the adsorption is dependent on hydrophobic interactions.

enter image description here

Does Fibronectin stick to the other proteins or rather to "free areas" of the surface?

It can stick to both as you can see from the table. However, its binding to hydrophobic materials like polystyrene and PVC is the most efficient.

which parameter is the best to vary?

Note that there will be a lot of free regions in the substrate. You cannot block all the sites unless you use a high concentration of fibronectin. So the best parameter to vary would be the concentration of fibronectin. You may also use fibronectin-BSA mixtures as their mutual binding is not that great (A stepwise method would be nonetheless better). You can also vary the adsorption time (see the figure below) but that is not as easily controllable as concentration. Since fibronectin does not bind to agar, you can also block some sites on your coverslip using molten agar (or agarose).

enter image description here


Reference:

Klebe, Robert J., Kevin L. Bentley, and Robert C. Schoen. "Adhesive substrates for fibronectin." Journal of cellular physiology 109.3 (1981): 481-488.

$\endgroup$
  • $\begingroup$ Thanks, really nice answer! Thanks also for the reference. So in my experiments (which is actually not exactly micro-contact printing) I varied Protein concentration/Incubation time already a lot. However, this does not yield the wanted result - Fibronectin will just adhere onto the first Protein (Laminins/Vitronectin etc), even tried BSA-blocking. Just out of curiosity: what is telling you, that there are lots of free areas on my substratum after adsorption? $\endgroup$ – AnatraIlDuck Sep 21 '15 at 13:07
  • $\begingroup$ @Vincent Well just an intuitive calculation. If surface area of the substrate is in the order of mm² and lets say the area occupied by each FN molecule is in the order of nm² (taking the length of FN as diameter of a circle). You can adsorb at least ~10⁶ molecules. So you are coating with a layer of lamins and then FN? Reducing the concentration should work. $\endgroup$ – WYSIWYG Sep 21 '15 at 16:58
  • $\begingroup$ Well I reduced the concentration, still FN seems to stick to "laminin"-areas. Sure, I will accept your answer. I'm just waiting one or two more days - perhaps someone had already a similar problem and another hint. $\endgroup$ – AnatraIlDuck Sep 21 '15 at 18:41

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.