No Polymerization...

EmRy2

I have a hydrogel solution with acrylamide, bis-acrylamide (both from Bio-rad), Va-044 from WAKO, PBS, and H20. I fixed my samples for 24 hrs in 4% PFA before placing them in the hydrogel solution for three days. Instead of using a desiccator, I have tried both Peanut oil alone as well as Peanut oil after removing the air in a vacuum from the hydrogel. However, both sets of gel have not polymerized after sitting in a 37 degree water bath for more than three hours. I have tried increasing the acrylamide by 1%, and that has not worked either. Any suggestions on where else I may have gone wrong?

MariaSuessmilch

Hi, I'll walk you through what I did, which worked fine:

Hydrogel solution: PREPARE ALL ON ICE and KEEP AT -20 degrees C. 4% acrylamide, 0.05% bis-acrylamide, 0.25% VA-044, 1x PBS, 4% PFA and distilled water.

(PFA is crucial in this step as this will bind to your proteins/nucleic acids, receptors etc. and then the acrylamide will form a bond with the PFA!)

Transcardial perfusion of mouse with PBS (20ml) then hydrogel solution (20ml), brain sat in hydrogel solution at 4 degrees for 3 days.

Added 15ml of vegetable oil to 50ml falcon containing hydrogel and brain. Placed in water bath at 37 degrees for 3 hours.

So points where it could have gone wrong:

No PFA in your hydrogel solution?

Keep everything on ice all the time? If not the hydrogel might have started to polymerize at RT.

Perfused with hydrogel solution? Did you leave it in hydrogel solution after?

I hope this helps! Let me know if you've got more question!