230
- After staining, remove the membrane and place into the destain
solution ( see Note 10 ). - Wash the membrane on rocking platform for 10 min, changing
solution every 5 min. - After destaining, wash the membrane with water and dry
completely. - Analyze the resulting bands via densitometry (Fig. 1 ). (We use
the ImageJ software for densitometry analyses.) - Add 2 mL of DMF to 60 mg of dry M-270 epoxy Dynabeads.
- Vortex the beads for 2 min and keep at 4 °C. The beads may
be stored indefi nitely at 4 °C. - Vortex the stock of beads for 1–2 min and transfer 200 μL of
beads into a microcentrifuge tube ( see Note 11 ). - Place tube on magnetic stand to separate beads from DMF for
1 min and discard the supernatant. - Wash the beads with 1 mL of sodium phosphate buffer: gently
resuspend the beads in the buffer by rotation and then place
tube on magnet for 1 min. - Discard supernatant and repeat step 5 for additional two times.
- Immediately, add 230 μL of sodium phosphate buffer to the
dry beads. - Add 10 μL of antibody (10 mg/mL stock concentration) to
the mixture and vortex for 15 s. - Add 120 μL of ammonium sulfate buffer to the mixture.
- Incubate the mixture at 37 °C for 16–24 h on a nutator.
- Following incubation, wash the beads four times with 1 mL of
PBS as described in step 5.
3.5 Immuno-
precipitation
Fig. 1 Filter trap analyses of insoluble protein aggregates. Twofold serial dilutions
of the soluble (SOL) and insoluble (INS) protein fractions were fi ltered through a
0.45 μM PVDF membrane and stained with Coomassie Blue solution. The labels
on the right indicate the different dilutions (D1–D4)Amit Shrestha et al.http://www.ebook3000.com