Secretome


Centrifuge TCA ppts 10min at 7KRPM (HS4 rotor)

Be sure buckets are properly attached and contain adapters

Run SDS PAGE
Stain gel

Make up 500 ml 0.1M ammonium acetate

Make up protein stock solutions for further ppt experiments

RNAse, DNase, Human albumin ~1mg/ml (in ammonium acetate)

Make 30 ml solution with 1 microgram/ml (1x) of each of the above

Dilute 2x, 4x, 8x

To 15 ml of each, add add small amount of charcoal and then 2ml 100% w/v TCA

Make up 20ml, 1% deoxycholate solution

To 15 ml of each, add 0.75ml DOC and then 2ml 100% w/v TCA

Mix, store overnight on ice (in cold room)

Centrifuge, dissolve pellets in 100 microliters cracking buffer, electrophorese