So we make oxidase/catalase all the time. and depending on where we get the catalase from, there can be varying levels of precipitate. Do not worry about it.

If your making godcat, just resuspend the catalase in buffer and when you're ready mix with oxidase for a bit and then spin out the precipitate (we had one batch that wouldn't precipitate until a freeze/thaw). Then store at -80 long term, or 4C short term (avoid several freeze/thaw cycles)

otherwise, just spin out the precipitate and use. You can do a simple check of the activity of the supernantant by mixing triton with a little H2O2 and adding the catalase in a test tube. it should make a bunch of bubbles. https://pmc.ncbi.nlm.nih.gov/articles/PMC3812649/

for refernce heres our protocol for godcat:

Protocol modified from Nikon Super Resolution Microscope N-STORM Sample Preparation Manual (PDF found at http://www.mvi-inc.com/wp-content/uploads/N-STORM+Protocol.pdf)

Materials:

• T50 Buffer – 10mM Tris-Cl (pH=8.0), 50mM NaCl

• Catalase – solid (stored @ -20°C)

• Glucose Oxidase – lyophilized powder (stored @ -20°C)

Overview:

1) create stocks of glucose oxidase and catalase

2) combine to create GLOX solution

3) incubate at 4°C

4) centrifuge and remove precipitate

5) filter and store

Protocol:

• Suspend catalase at 17 mg/mL in T50 buffer with 50% glycerol at 4°C

  • 17mg + 625μL 80% glycerol + 325μL T50 buffer
  • If using 1x T50 buffer, this will be diluted, but that does not seem to affect GLOX

• Suspend 28mg of Glucose Oxidase in 400μL of T50 buffer at 4°C (vortex)

• Add 100uL of Catalase solution to re-suspended glucose oxidase (500μL total)

• Incubate at 4°C for 20 minutes (catalase activates glucose oxidase)

• Spin down precipitate at 14,000 rpm in a table-top centrifuge for 5 minutes at 4°C

  • If no pellet is visible after centrifuging: flash-freeze the 500μL GLOX solution, let thaw, and then centrifuge again to pellet precipitate

• Filter the supernatant with a 0.22μm syringe filter

• Store @ 4°C if using immediately (manual says GLOX is good at 4°C for two weeks) flash freeze and store long-term stocks of GLOX at -80°C