I am interested in doing a western blot to detect expression of focal adhesion proteins in cells that are encapsulated in alginate hydrogels. The problem seems to be the infiltration of e.g. SDS or RIPA inside the gels. Does anybody have a positive experience with this?
Thanks,
Taimoor
hi Taimoor
Hope you are doing good? Im assuming you are using calcium cross-linked alginate hydrogels? If that is the case then an easy way out of this could be to just dissolve the gels using high concentration of EDTA while keeping it at 4 C and then just centrifuge the solution to separate your cells from the gel. If you are doing just the level of expression then it should work, but if its phospho-proteins then you may lose the signal.. I hope this helps! :)
Hi Taimor,
EDTA will work to disrupt your gels but if you want someting smoother, you may try citric acid. Good luck.
Thank you for your replies. However, I fear the expression of my proteins of interest will be lost if I take them out of the gel by EDTA or citrate.
Hi Taimoor,
When I've run Westerns for p-Smads in the past for MSCs in alginate, I just transferred the entire gel directly into a roughly equal volume of 2x RIPA buffer in a microcentrifuge tube and then disrupted the gel by sonicating with a probe sonicator. Because the alginate is only physically crosslinked by the calcium ions, it will be reversibly disrupted by the sonication. This will also lyse the cells and release your protein of interest. If you then spin the samples at max speed at 4 C for 10-15 minutes, you will separate your lysate from the gel and other cellular debris. You can pipet off the supernatant and run your Westerns with that.
Hope this helps,
Chris
Hi Taimoor,
I used PEG-Fibrinogen hydrogel, but I think is the same thing. We use snap freeze the structure in liquid nitrogen, that keeping the gel in liquid nitrogen into a mortar we grind the samples witha pestle obtaining more or less fine powder that we resuspend in RIPA for protein extaction using standard protocol.
I hope this will help you.
Good luck
Hi Hui Zhu , The protocol referenced above was published in this article: Article Presentation of BMP-2 Mimicking Peptides in 3D Hydrogels Dir...
For that study, I chose to sonicate rather than chelate out the calcium with EDTA because I wanted to capture a snapshot of the Smad signaling response shortly after encapsulation. If I were to dissolve the gels, the specific phosphorylation signal I was looking for could have been lost as the gels were dissociating. So, if you are interested in looking at signaling cascades that may turn on and off in a matter of minutes, I would recommend the sonication approach. Additionally, if you are interested in assaying secreted proteins (ECM or signaling factors) that could be associated with the gel, the sonication approach would be my first choice. If you are just looking at relative amounts of a particular intracellular or transmembrane protein that is not expected to change over the time scale of gel dissolution, then the EDTA approach should also work.
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